Publications
Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.
Modification of acyl carrier proteins (ACP) or domains by the covalent binding of a 4′-phosphopantetheine (4′-PP) moiety is a fundamental condition for activation of fatty acid synthases (FASes) and polyketide synthases (PKSes). Binding of 4′-PP is mediated by 4′ phosphopantetheinyl transfersases (PPTases). Mycobacterium tuberculosis (Mtb) possesses two essential PPTases: acyl carrier protein synthase (Mtb AcpS), which activates the multidomain fatty acid synthase I (FAS I), and Mtb PptT, an Sfp-type broad spectrum PPTase that activates PKSes. To date, it has not been determined which of the two Mtb PPTases, AcpS or PptT, activates the meromycolate extension ACP, Mtb AcpM, en route to the production of mycolic acids, the main components of the mycobacterial cell wall. In this study, we tested the enzymatic activation of a highly purified Mtb apo-AcpM to Mtb holo-AcpM by either Mtb PptT or Mtb AcpS. By using SDS-PAGE band shift assay and mass spectrometry analysis, we found that Mtb PptT is the PPTase that activates Mtb AcpM. We measured the catalytic activity of Mtb PptT toward CoA, using an activation assay of a blue pigment synthase, BpsA (a nonribosomal peptide synthase, NRPS). BpsA activation by Mtb PptT was inhibited by Mtb apo-AcpM through competition for CoA, in accord with Mtb AcpM activation. A structural model of the putative interaction between Mtb PptT and Mtb AcpM suggests that both hydrophobic and electrostatic interactions stabilize this complex. To conclude, activation of Mtb AcpM by Mtb PptT reveals a potential target of the multistep mycolic acid biosynthesis.
The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from the thermophile Thermoanaerobacter hrockii (TbADH), the mesophilic bacterium Clostridium heijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Three chimeras have been constructed. In the first chimera, the cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH [chimera X21(TCT)]. This domain exchange significantly destabilized the parent thermophilic enzyme (AJi/2 = -18 °C). The reverse exchange in CbADH [chimera X22(TCT)]. however, had little effect on the thermal stability of the parent mesophilic protein. Furthermore, substituting the cofactor-binding domain of TbADH with the homologous domain of EhADHl [chimera X23(TET)] substantially reduced the thermal stability of the thermophilic ADH (ΔT1/2 = -51 °C) and impeded the oligomerization of the enzyme. All three chimeric proteins and one of their site-directed mutants were crystallized, and their three-dimensional (3D) structures were determined. Comparison of the 3D structures of the chimeras and the chimeric mutant with the structures of their parent ADHs showed no significant changes to their Cα chains, suggesting that the difference in the thermal stability of the three parent ADHs and their chimeric mutants could be due to a limited number of substitutions located at strategic positions, mainly at the oligomerization interfaces. Indeed, stabilization of the chimeras was achieved, to a significant extent, either by introduction of a proline residue at a strategic position in the major horse liver ADH-type dimerization interface (ΔT1/2 = 35 °C) or by introduction of intersubunit electrostatic interactions (Δ T 1/2 = 6 °C).
We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.
Analysis of the three-dimensional structures of two closely related thermophilic and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Entamoeba histolytica (EhADH1) and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro275) at the center of the dimerization interface might be crucial for maintaining the thermal stability of TbADH. To assess the contribution of Pro275 to the thermal stability of the ADHs, we applied site-directed mutagenesis to replace Asp275 of EhADH1 with Pro (D275P-EhADH1) and conversely Pro275 of TbADH with Asp (P275D-TbADH). The results indicate that replacing Asp275 with Pro significantly enhances the thermal stability of EhADH1 (ΔT1/2 ≤ + 10°C), whereas the reverse mutation in the thermophilic TbADH (P275D-TbADH) reduces the thermostability of the enzyme (ΔT1/2 ≤ -18.8°C). Analysis of the crystal structures of the thermostabilized mutant D275P-EhADH1 and the thermocompromised mutant P275D-TbADH suggest that a proline residue at position 275 thermostabilized the enzymes by reducing flexibility and by reinforcing hydrophobic interactions at the dimer-dimer interface of the tetrameric ADHs.
Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: ΔT1/260 min = +8°C (temperature of 50% inactivation after incubation for 60 min), ΔT1/2CD = +11.5°C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30° and 98°C). A His100 → Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region.
The structure of the apo form of alcohol dehydrogenase from a single-cell eukaryotic source, Entamoeba histolytica, has been determined at 1.8 Å. To date, bacterial and archeal alcohol dehydrogenases, which are biologically active as tetramers, have crystallized with tetramers in the asymmetric unit. However, the current structure has one independent dimer per asymmetric unit and the full tetramer is generated by application of the crystallographic twofold symmetry element. This structure reveals that many of the crystallization and cryoprotection components, such as cacodylate, ethylene glycol, zinc ions and acetate, have been incorporated. These crystallization solution elements are found within the molecule and at the packing interfaces as an integral part of the three-dimensional arrangements of the tetramers. In addition, an unexpected modification of aspartic acid to O-carboxysulfanyl-4-oxo-L-homoserine was found at residue 245.
The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.
Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP+/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.
Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme. We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 Å, using the molecular replacement method. The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ∼222-point symmetry. The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria. The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (kcat = 4.5 sec -1; Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate. The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path. The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule.
Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.
The tetrameric NADP+-dependent secondary alcohol dehydrogenase from Entamoeba histolytica has been crystallized in its apo form. The crystals belong to space group C2221, with unit-cell parameters a = 76.89, b = 234.24, c = 96.24 Å, and diffract to 1.9 Å at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function shows that the crystals contain one dimer per asymmetric unit.
Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) catalyzes the reversible oxidation of secondary alcohols to the corresponding ketones using NADP+ as the cofactor. The active site of the enzyme contains a zinc ion that is tetrahedrally coordinated by four protein residues. The enzymatic reaction leads to the formation of a ternary enzyme-cofactor-substrate complex; and catalytic hydride ion transfer is believed to take place directly between the substrate and cofactor at the ternary complex. Although crystallographic data of TbADH and other alcohol dehydrogenases as well as their complexes are available, their mode of action remains to be determined. It is firmly established that the zinc ion is essential for catalysis. However, there is no clear agreement about the coordination environment of the metal ion and the competent reaction intermediates during catalysis. We used a combination of X-ray absorption, circular dichroism (CD), and fluorescence spectroscopy, together with structural analysis and modeling studies, to investigate the ternary complexes of TbADH that are bound to a transition-state analogue inhibitor. Our structural and spectroscopic studies indicated that the coordination sphere of the catalytic zinc site in TbADH undergoes conformational changes when it binds the inhibitor and forms a pentacoordinated complex at the zinc ion. These studies provide the first active site structure of bacterial ADH bound to a substrate analogue. Here, we suggest the active site structure of the central intermediate complex and, more specifically, propose the substrate-binding site in TbADH.
Viral persistence and molecular latency are characteristic of infection by the human cytomegalovirus (HCMV). Using the murine cytomegalovirus (MCMV) as a model for human infection, a quantitative-competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify MCMV-DNA in the salivary glands of infected mice. The QC-PCR detected high numbers of MCMV DNA copies in the absence of infectious virus. By comparing the DNA content and the results obtained from a standard semiquantitative plaque assay, it is concluded that 1 plaque-forming unit (pfu) is the equivalent of approximately 1500 viral genomes. By day 42-post infection (pi) 4 x 103 copies of DNA/1 mg tissue were sufficient to reactivate infectious virions after cyclophosphamide immunosupression. By day 90 pi, however, when the DNA load was decreased to 2, reactivation was not observed. These results indicate that viral reactivation will occur when the number of infectious DNA copies is equivalent about 2-3 pfu. This quantitative test may therefore help to detect CMV and the risk of reactivation in immunosupressed patients. (C) 2000 Elsevier Science B.V.
Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30 degrees in their melting temperatures. The crystal structures of CBADH and TBADH in their hole-enzyme form have been determined at a resolution of 2.05 and 2.5 Angstrom, respectively. Comparison of these two very similar structures (RMS difference in C alpha = 0.8 Angstrom) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, "charged-neutral" hydrogen bonds, and prolines as well as improved stability of alpha-helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.
The essential role played by the thymus in the development of the immune response was well documented in many publications. These findings prompted a long series of studies devised to define the factors produced and secreted by thymus cells, which are involved in the development and nature of immunological responsiveness. First experiments done with crude thymus extracts were followed by isolation of purified products and finally by chemical characterization and synthesis of immunologically active thymus- derived peptides. In this article we review the various thymic hormones and factors described, that is, thymosin fractions 5, the thymosins, prothymosin α, thymulin (FTS-Zn), thymopoietin, thymostimulin (TP-1), Thymic humoral factor (THF), and THF-γ2. Studies demonstrating the activity of the various thymic factors in increasing the immunocompetence potential in both in vitro and in vivo conditions are discussed. The immunostimulatory potential of thymic factors was also investigated in experimental models where beneficial therapeutic effects were sought in a situation of immunological malfunction. The last part of the review is dedicated to clinical trials with thymic factors that revealed improvement in the immunocompetence potential in cases of immunodeficiencies, viral infections, and cancer and its correlation with therapeutic effectiveness. It seems that more research is required in order to better define conditions for the use of thymic factors in immunotherapy.
Immunotherapy with the immunomodulating thymic humoral factor-γ2 (THF-γ2) octapeptide, combined with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy, will be used for enhancing host immune response to arrest pulmonary metastases of a B16-F10.9 melanoma tumor. In this experimental model of pulmonary metastasis, the highly metastatic B16-F10.9 melanoma tumor cells (2 × 105) were inoculated into the footpad of mice to form a primary tumor. The tumor-bearing leg was surgically removed on reaching the size of 5.5 mm, which resulted in the appearance of metastases in the lungs of the animals. After tumor excision, mice were treated intraperitoneally with a single dose of BCNU (20 or 35 mg/kg) followed by a series of intraperitoneal THF-γ2 injections (1 µg’0.5 ml’injection). Relative to untreated mice and those receiving chemotherapy alone, the antitumor action of the combined THF-γ2 chemoimmunotherapy protocol was significantly augmented according to the following in vivo parameters: (a) decreased postsurgical spontaneous metastatic burden; (b) prolonged survival time; (c) increased resistance to tumor cell challenge, and (d) massive infiltration of lymphocytes, polymorphonuclear cells, and macrophages in the lung tissue. The THF-γ2 immunotherapy also prevented a decrease in lymphocyte reactivity, otherwise induced by the tumor’BCNU chemotherapy. THF-γ2 immunotherapy resulted in restoration of the response to Lipopolysaccharide mitogenic stimulation and the allogeneic response. Our data suggest that postoperative THF-γ2 immunotherapy could be a valuable adjunct to anticancer chemotherapy as a treatment for metastatic arrest of melanoma tumor.
A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala- 347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a β-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of α-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.
We have determined the X-ray structures of the NADP(H)-dependent alcohol dehydrogenase of Clostridium beijerinckii (CBADH) in the apo and holo-enzyme forms at 2.15 Å and 2.05 Å resolution, respectively, and of the holo-alcohol dehydrogenase of Thermoanaerobacter brockii (TBADH) at 2.5 Å. These are the first structures of prokaryotic alcohol dehydrogenase to be determined as well as that of the first NADP(H)-dependent alcohol dehydrogenase. CBADH and TBADH have 75% sequence identity and very similar three-dimensional structures. Both are tetramers of 222 symmetry. The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain. These are separated by a deep cleft at the bottom of which a single zinc atom is bound in the catalytic site. The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates. The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft thus leaving it accessible from the surface of the tetramer. The tetramer encloses a large internal cavity with a positive surface potential. A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer. The specificity of the two bacterial alcohol dehydrogenases towards NADP(H) is determined by residues Gly198, Ser199, Arg200 and Tyr218, with the latter three making hydrogen bonds with the 2'-phosphate oxygen atoms of the cofactor. Upon NADP(H) binding to CBADH, Tyr218 undergoes a rotation of approximately 120°about χ1 which facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygen atoms. In apo-CBADH the catalytic zinc is tetracoordinated by side-chains of residues Cys37, His59, Asp150 and Glu60; in holo-CBADH, Glu60 is retracted from zinc in three of the four monomers, whereas in holo-TBADH, Glu60 does not participate in Zn coordination. In both holo-enzymes, but not in the apo-enzyme, residues Ser39 and Ser113 are in the second coordination sphere of the catalytic zinc. The carboxyl group of Asp150 is oriented with respect to the active carbon of NADP(H) so as to form hydrogen bonds with both pro-S and pro-R hydrogen atoms.
Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophile Clostridium beijerinckii (CBADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26°C in thermal stability. To explore the role of linear segments of these similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed in Escherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.
Proteins play a pivotal role in thermophily bacteria. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed in E. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme.
Synthesis of two chimeric peptides composed of tuftsin and thymic humoral factor-γ2 (THF-γ2) conjugates was accomplished. Our goal was the generation of novel immunomodulators. Initially, we demonstrate an IL-6 inducing activity of the phagocytic cells stimulant, tuftsin, on murine macrophages. This activity was documented only in the presence of antigen, either KLH or lysozyme. The augmentation was dose dependent, with optimal activity at a concentration of 200 and 20 nM, respectively. The chimeric peptides, either H2N-tuftsin-THF-γ2-OH or H2N-THF-γ2-tuftsin-OH, were also evaluated in the IL-6 system in the presence of the more potent antigen, KLH. The IL-6 inducing effect was maintained, although maximal activity appeared only at a concentration an order of magnitude greater than that of tuftsin. The chimeric peptides were further tested in an assay evaluating enhancement in murine bone marrow myeloid colony formation, a system in which THF-γ2, a T cell stimulant, has an established beneficial effect. The compounds were found to be inactive at the 25-200 ng/ml (14-112 nM) concentration range evaluated. Finally, the chimeric peptides were tested in a combined macrophages-T cells assay, i.e, antigen presentation, in which H2N-tuftsin-THF-γ2-OH was found to be more active than either parent peptide, thus representing a possible therapeutic agent.
The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol- disulfide exchange reaction and with 2[14C]iodoacetic acid, via S- alkylation. The respective reactions were monitored by electron paramagnetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR date suggested a distance of ≤ 10 Å between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75°C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.
The active-site metal ion and the associated ligand amino acids in the NADP-linked, tetrameric enzyme Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized by atomic absorption spectroscopy analysis and site-directed mutagenesis. Our preliminary results indicating the presence of a catalytic zinc and the absence of a structural metal ion in TBADH (Peretz & Burstein. 1989. Biochemistry 28:6549-6555) were verified. To determine the role of the putative active-site zinc, we investigated whether exchanging the zinc for other metal ions would affect the structural and/or the enzymatic properties of the enzyme. Substituting various metal ions for zinc either enhanced or diminished enzymatic activity, as follows: Mn2+ (240%); Co2+ (130%); Cd2+ (20%); Cu2+ or V3+ (
An optimal therapeutic regimen against primary CMV salivary-gland infection has not yet been developed. We used a murine CMV (MCMV) model system to assess the ability of combined thymic humoral factor THF-gamma 2 immunotherapy and ganciclovir (GCV) antiviral chemotherapy to eliminate detectable viral DNA from salivary glands of infected animals. Mice in different experimental groups were inoculated intraperitoneally with MCMV, treated, and then sacrificed either 2 weeks or 3 months later. To amplify and detect MCMV DNA in infected salivary-gland tissue, we developed a sensitive polymerase chain reaction (PCR) using a glycoprotein B gene primer pair that amplifies a 356 bp segment. During the acute phase of the infection, the detection of high titers of infectious virus in the salivary glands correlated with a strong PCR amplification signal. Although active virions could not be recovered from untreated animals 3 months after viral inoculation, the PCR assay detected a latent MCMV genome. Treatment with either GCV alone or THF-gamma 2 alone had little or no effect on the presence of MCMV DNA. By contrast, combined treatment with THF-gamma 2 and GCV significantly reduced the amount of salivary-gland MCMV DNA to below the limit of PCR detection. The results presented here, and experimental data from previous MCMV research in our laboratories, imply that elimination of the virus from the salivary glands could be due in part to THF-gamma 2 restoration of the various MCMV-suppressed cell mediated immune-responses Combining THF-gamma 2 immunotherapy and GCV antiviral chemotherapy may-be an important step toward an effective therapeutic regimen that has the potential to prevent the establishment of viral latency ensuing from primary MCMV salivary-gland infection.
Previous research in our laboratories has shown that the immunoregulatory octapeptide. THF-γ2, potentiates the efficacy of anticancer chemotherapy in experimental animal models of local plasmacytoma and repairs drug-induced defects in immunocompetence. The highly metastatic, murine D122 lung carcinoma model has been shown to be useful for evaluating the efficacy of experimental antimetastatic therapeutic modalities. The goal of the present study was to determine whether intranasal thymic humoral factor-γ2 (THF-γ2) immunotherapy, after a single dose of chemotherapy, could inhibit the development of lung metastases, restore immunocompetence, and increase survival in syngeneic C57BL/6 mice bearing highly metastatic Lewis lung carcinoma (D122) solid footpad tumors. Relative to untreated mice and those receiving chemotherapy alone, mice receiving combined chemoimmunotherapy showed the following significant differences: (a) decreased lung metastatic load as assessed by lung weight, (b) prolonged survival time, (c) massive infiltration of lymphoid cells in the lungs, and (d) restoration of impaired immune parameters to normal values in melphalan-treated mice. THF-γ2 prevented tumor emboli from colonizing the target tissue, probably by inducing expansion of the lymphoid cell compartment. When used as an adjunct to anticancer chemotherapy, intranasal THF-γ2 immunotherapy is a simple and safe treatment modality that seems to be promising for inhibiting lung metastases.
Two tetrameric NADP+-dependent bacterial secondary alcohol dehydrogenases have been crystallized in the apo- and the holoenzyme forms. Crystals of the holo-enzyme from the mesophilic Clostridium beijerinckii (NCBAD) belong to space group P212121 with unit-cell dimensions a = 90.5, b= 127.9, c=151.4 A. Crystals of the apo-enzyme (CBAD) belong to the same space group with unit-cell dimensions a = 80.4, b= 102.3, c= 193.5 Å. Crystals of the holo-enzyme from the thermophilic Thermoanaerobium brockii (NTBAD) belong to space group P61(5) (a= b = 80.6, c = 400.7 Å). Crystals of the apo-form of TBAD (point mutant G198D) belong to space group P21 with cell dimensions a= 123.0, b = 84.8, c=160.4 Å β= 99.5°. Crystals of CBAD, NCBAD and NTBAD contain one tetramer per asymmetric unit. They diffract to 2.0 Å resolution at liquid nitrogen temperature. Crystals of TBAD(G198D) have two tetramers per asymmetric unit and diffract to 2.7 Å at 276 K. Self-rotation analysis shows that both enzymes are tetramers of 222 symmetry.
Recent findings have demonstrated that the GnRH gene is expressed in the mammary gland of pregnant and lactating rats but not of virgin rats. Indeed, significant concentrations of biologically active GnRH have been found in milk of human, cow, sheep and rat, We have, therefore, looked for expression of the GnRH receptor in the rat mammary gland. By reverse transcription (RT)-PCR amplification, we have demonstrated the presence of GnRH receptor mRNA in mammary gland samples derived from virgin, pregnant and lactating rats. The GnRH receptor transcript cloned from the mammary gland was sequenced and found to have an identical coding region to the one cloned from the pituitary gland. In addition, we have found that the mammary gland, as the pituitary gland, contains at least two transcripts having the same coding region but different 5' non-coding regions, Binding studies, however, could demonstrate only low-affinity binding sites. These results, therefore, suggest that the regulation of the GnRH receptor occurs posttranscriptionally rather than at the level of transcription.
In mice bearing immunogenic tumors, adding thymic humoral factor-γ2 (THF- γ2)1 immunotherapy as an adjunct to anticancer chemotherapeutic regimens not only potentiates the antitumor activity of each drug but also repairs tumor/chemotherapy-induced damage to T-cell populations and functions. The Lewis lung carcinoma (3LL) is a weakly immunogenic, highly metastatic tumor in C57BL/6 mice. To investigate whether the immunoregulatory octapeptide is also effective against a tumor that does not elicit an antitumor immune response, we assessed the effect of combination THF-γ2 immunotherapy and chemotherapy in 3LL-bearing mice. The results indicate that THF-γ2 combined with either Melphalan or 5-Fluorouracil was more effective in reducing metastatic load than either chemotherapeutic drug alone and was characterized by massive infiltration of lymphatic cells. The combined chemoimmunotherapy treatment also prolonged the survival time in all treated animals and repaired T-cell defects and impaired in vitro cellular immune response parameters, induced either by the tumor or by chemotherapy. THF-γ2 immunotherapy reversed the decrease in the number of bone-marrow myeloid colonies (GM-CFU) induced by chemotherapy treatment of tumor-bearing mice, supporting the hypothesis that THF-γ2 directly stimulates the proliferation of myeloid stem cells. The overall results imply, that when administered as an adjunct to chemotherapy, THF-γ2 immunotherapy is equally effective against immunogenic and nonimmunogenic tumors.
A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokine-deprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.
Murine CMV (MCMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV (HCMV) infection. MCMV causes T cell immune impairment in the infected mice, manifested by suppressed responses to T cell mitogens and a profound reduction of Con A induced IL-2 production. Thymic humoral factor (THF-gamma 2) is an octapeptide which was first isolated from calf thymus, characterized and chemically synthesized. This peptide has been shown to have immunoregulatory effects in various systems. Systemic treatment of MCMV-infected mice with THF-gamma 2 resulted in the enhancement of protective efficacy of MCMV immune spleen cells and the reconstitution of mitogenic responses and IL-2 secretion.
We have recently found that the inhibitor of plasmacytoma cell growth, restrictin-P, is a stroma derived activin A and that it is an antagonist of interleukin-6 and interleukin-11. The present study was aimed at determining the mode by which this cytokine kills its target cells. On addition of the cytokine there was little or no net increase in cell number, depending on the specific target cells. All plasmacytoma cell lines tested exhibited a similar time dependent inhibition of DNA synthesis and a G0/G1 shift in the cell cycle. Electron microscope examination revealed classical apoptotic features i.e. chromatin condensation and membrane blebbing. DNA fragmentation, measured qualitatively and quantitatively, occurred in all cytokine treated plasmacytoma cell lines. Bovine activin A had an identical capacity to reduce cell viability, to induce G0/G1 shift and to cause DNA fragmentation. X-ray microanalysis of intracellular ions revealed an increase in calcium ions, following exposure of plasmacytoma cells to restrictin-P, accompanied by a decrease in phosphor ions. The cytotoxicity of the inhibitor was augmented in an additive manner by cycloheximide (CHX) indicating that the process did not require de novo protein synthesis. This study thus shows that restrictin-P/stromal activin A kills its target cells by inducing apoptosis. This effect was mediated by subnanogram concentrations and therefore may represent one physiological function of this pleiotropic cytokine.
The high concentration of gonadotropin-releasing hormone (GnRH) in milk of several species implies that the mammary gland is either a site of synthesis for this neuropeptide or that it is efficiently concentrated from plasma by this organ. By PCR amplification of mammary gland cDNA, we have demonstrated expression of the mRNA for GnRH. The GnRH mRNA was present in the mammary gland of pregnant and lactating rats but not of virgin rats, implying that expression of the GnRH gene is activated during pregnancy, probably by prolactin. In contrast, actin mRNA was evident in all the preparations of mammary glands. Since GnRH is also known to be synthesized by the placenta, it is likely that the placenta and the mammary gland are complementary units by which the mother exercises control over the development and the metabolism of the infant during pregnancy as well as after parturition. In addition, GnRH synthesized by the mammary gland may also affect the mother by a paracrine and/or an endocrine mechanism.
An unidentified 30 kDa protein frequently copurifies with human glutathione S-transferases from S-hexyl-glutathione affinity matrices. Application of two-step sequential affinity chromatographic methods yielded a homogeneous preparation of that protein from human liver specimens. The protein was digested with Achromobacter protease I, and sequences of peptides resolved by h.p.l.c. showed a high degree of identity with those of rat mitochondrial Δ3,Δ2-enoyl-CoA isomerase. The human protein also exhibited catalytic activity with Δ3-cis-octenyl CoA as a substrate. Thus it is identified as liver Δ3,Δ2-enoyl-CoA isomerase.
Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. The contribution of the different spleen cell subsets in conferring curative immunocytotherapy to fatally MCMV-infected immuno-suppressed mice was assessed using adoptive immunotherapy. It was found that the efficacy of passively transferred immune spleen cells is dose dependent and that the therapeutic effect can be enhanced considerably by treating donor mice with thymic humoral factor (THF-γ2). Polymerase chain reaction (PCR) of the donor spleen population was negative, indicating that no MCMV-DNA was transferred with the immune cells. Analysis of the donor mice after THF-γ2 treatment showed increased levels of CMV-neutralizing antibodies, while enhancement of natural killer (NK) activity was transient and lasted only during the early phase of the infection. FACS analysis demonstrated that treatment with THF-γ2 restored the size of both cell subsets CD4+ and CD8+ that were decreased following MCMV infection. It is shown that both CD4+ and CD8+ T-cell subsets participate in controlling the development of the fatal disease in MCMV-infected immunosuppressed recipients. It is suggested that the enhancement of the immunocompetence of both populations of spleen cells from treated donors is mediated in part by the restoration of Interleukin-2 (IL-2) production by THF-γ2.
The abundant hydrophobic, proline-glutamine, and histidine-rich (over 90%) amelogenins constitute the major class of proteins in forming extracellular enamel matrix. These are thought to play a major role in the structural organization and mineralization of developing enamel. The present report describes the successful sequencing of the major human amelogenin protein, by use of both Edman degradation and cDNA sequencing. When Edman degradation was used, over 75% of the primary structure of the protein was determined. This sequence was supplemented with cDNA sequencing studies, which revealed the predicted sequence of this protein. Together, they provide the complete sequence of an important human enamel protein. The information complements recent studies on bovine and human amelogenin genes. A comparison between the present results and the protein sequences predicted from the corresponding human amelogenin genomic coding regions and that of cDNA sequences of other species is described.
Class A and class B NAD(H):NADP(H) coenzyme‐dependent dehydrogenases distinguish between the diastereotopic hydrogens pro‐R and pro‐S at position 4 of the cofactor. We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc‐containing, NADP‐linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD). Using proton NMK spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro‐R hydrogen from the pyridine 4 position of the reduced coenzyme. This stereospecificity is stable over (a) a broad range of temperatures up to 70° C. (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates. Although NAD + is not an effective coenzyme for TBAD, NADP + and its synthetic analogs, 3‐acetqlpyridine‐ADP + and thio‐NADP +, can be used successfully.
A bacterial thermophilic alcohol dehydrogenase which is stable and active at 85°C, has been crystallized by vapor diffusion from solutions of polyethylene glycol. A monoclinic crystal form diffracts to 2·8 Å resolution and belongs to space group C2 with unit cell dimensions a = 139·0 Å, b = 137·4 Å, c = 0·9 Å and β = 93·23°. The asymmetric unit contains four molecules which exhibit 222 point symmetry. A second crystal form is orthorhombic, space group P21212 with unit cell dimensions a = 168·0 Å, b = 123·0 Å, c = 80·0 Å, and it diffracts to 3·2 Å resolution.
The effect of the thymic hormone THF-γ2 on committed stem cells of bone marrow (BM) origin was determined using the myeloid progenitor cell clonal assay. Preincubation of normal BM cells with THF-γ2 for 1 hour or 18 hours caused a 2- to 6-fold increase in the number of myeloid colonies in the presence of suboptimal concentrations of colony-stimulating factor (CSF). The optimal dose of THF-γ2 causing this enhancement was in the range of 25 to 100 ng/mL. THF-γ2 was not able to replace CSF as an inducer in these experiments. THF-γ2 neither induced IL-6 activity upon 24-hour incubation with bone marrow cells nor enhanced LPS-induced IL-6 secretion by bone marrow cells in vitro. Neonatal thymectomy (NTx) of Balb/c mice caused a decrease in myeloid progenitors, which was repaired by serial injections of THF-γ2. The repair of the stem cell compartment in the bone marrow correlated with an increased percentage of Thy1+ cells in the spleen of THF-γ2-treated NTx mice. These findings indicate that THF-γ2 is able to regulate committed stem cell functions in the bone marrow of immune-deprived NTx and of normal mice.
The extracellular elastase (33 kDa) or Pseudomonas aeruginosa is synthesized as a 53.6 kDa preproenzyme containing a long, N-terminal propeptide. The free propeptide and the elastase precursor generated upon propeptide removal were isolated from P. aeruginosa cells and subjected to N-terminal amino acid sequence analysis. The results identified Ala-174 and Ala+1 as the amino terminal residues of the propeptide and the elastase precursor, respectively, indicating that: (1) the signal peptide consists of 23 amino acid residues and its molecular weight is 2.4 kDa, (2) the propeptide contains 174 amino acid residues and is of 18.1 kDa molecular weight, and (3) no additional N-terminal proteolytic cleavage is required for elastase maturation.
Thymus humoral factor-gamma-2 (THF-gamma-2), an octapeptide important for T-lymphocyte regulation, was assessed for its effect on the in vitro growth of human hematopoietic progenitor cells. This was achieved using a recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF)-stimulated myeloid cell colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) assay as well as a recombinant erythropoietin (rEpo)-stimulated erythroid burst formation (erythroid burst-forming units, BFU-E) assay. Cells were obtained from bone marrow (BM) and peripheral blood (PB) of normal healthy donors and from patients with suppressed bone marrows. The latter group included aplastic anemia, leukemia, and lymphoma patients and patients with solid tumors who responded to intensive chemotherapy with significant pancytopenia. THF-gamma-2 significantly enhanced normal BM and PB GM-CFC and PB BFU-E by 2- to 2.5-fold. This effect was totally dependent on the presence of the respective growth factors, that is, rGM-CSF or rEpo, and was specifically reversed by an anti-THF-gamma-2 antiserum). Furthermore, although THF-gamma-2-induced enhancement of GM-CFC colony formation was not affected by lymphocyte or monocyte depletion, the augmenting effect of the peptide on BFU-E was completely abrogated in the absence of lymphocytes. THF-gamma-2-induced augmented growth of progenitor cells derived from severely suppressed marrows was minimal. However, cells from moderately neutropenic patients with leukemia in remission or with lymphoma under chemotherapy responded to the peptide similarly to cells from normal donors. These results suggest a stimulatory role for THF-gamma-2 on human myeloid and erythroid hematopoietic progenitor cells. They also suggest the lymphocyte dependence of BFU-E enhancement and lymphocyte independence of GM-CFC stimulation by THF-gamma-2. In the former case the thymus-derived peptide may act through the induction of certain erythroid-enhancing lymphokin
Ascorbate peroxidase active component (APAC) was purified and characterized in Synechococcus PCC 9742 (R2) cells. APAC was isolated from freshly harvested cells, by ion exchange chromatography on DEAE cellulose, ultrafiltration through a 3000 dalton cut off filter and high pressure liquid chromatography through a reversed phase C-18 column. APAC was found to be extremely stable to harsh treatments of boiling water for 30 min, acidification to pH 2.0 and proteolytic digestion. A close correlation between activity and iron content of APAC was observed throughout the purification steps. E.S.R. spectrum of APAC showed a resonance line at g = 4.3 in the oxidized from. Peroxide reduction by ascorbate decreased the E.S.R. signal, which reappeared upon reoxidation by H2O2. The affinities of APAC to H2O2 and ascorbate were high (0.38 mM and 0.2 mM, respectively). Amino acid composition analysis of APAC revealed the presence of glutamic acid: glycine: cysteine residues at 2: 1: 1 ratio.
We reported previously that treatment of mice bearing MOPC—315 plasmacytoma with the drugs L—PAM (phenylalanine mustard) or S—FU (5—fluorouradl), in combination with low doses of THF—γ2, was more effective in increasing their survival time than treatment with the drug alone. We show here that in the combined treatment using a single injection of S—FU followed by multiple (8–15) injections of THF—γ2, the megadoses were more effective than the low doses in increasing the survival time of MOPC—315 tumor—bearing mice. On the other hand, in combination with L—PAM, both low and high doses of THF—γ2 were equally effective. The need for high doses of THF—γ2, when used in combination with 5—FU, could be due to the fact that 5—FU acts as a “non—immuno—modulating” drug and has to be used at a high, immunosuppressive dose.
The complete amino acid sequence of recombinant human Cu‐Zn superoxide dismutase (CuZnSOD) is presented. The S‐carboxymethylated protein was cleaved at lysine residues (with Achromobacter protease I) to provide a set of nine non‐overlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by cleavage at glutamic acid residues (with S. aureus V8 protease) and at arginine (with clostripain). The recombinant protein contains a single disulfide bond between cysteine residues 57 and 146. The primary sequence of recombinant human CuZnSOD is identical to that predicted by its cDNA sequence.
A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor γ2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 μM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA) in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF.
BALB/c mice cured of large MOPC‐315 plasmacytomas by melphalan remain deficient in their spleen T‐cell functions. This was manifested by impairment of the allogeneic and the antibody responses in vitro to SRBC and in decreased numbers of T‐cells including their subsets CD4 and CD8. IL‐2 production and specific cytotoxicity against MOPC‐315 tumor cells were, on the other hand, maintained. Treatment of these cured mice by in‐vivo administration of THF‐γ2, an octapeptide from calf thymus, repaired these deficits. This was evidenced by in vitro tests with spleen cells which manifested an increased allogeneic response and elevated generation of primary antibody response, restoration of T‐cell subpopulations to normal and an enhanced IL‐2 production above normal levels. The potential use of THF‐γ2 as supportive therapy in cancer treatment is suggested.
The effect of a synthetic thymic hormone, THF-γ2, on the anti-tumor activity of spleen cells was studied in mice immunized against the RPC-5 tumor. Following two courses of the THF-γ2 treatment, the mean RPC-5 specific cytotoxic response of immune spleen cells was significantly increased when compared to normal cells (P
Phytotoxins produced by a strain of Verticillium dahliae pathogenic on potato have been purified from culture fluids and xylem extracts of potato stems infected by the fungus. The toxins were very similar to each other in molecular mass (approximately 1000 D), HPLC profile, amino acid composition, biological activity and antigenic cross-reactivity. Visible Verticillium wilt symptoms were observed when either peptide, in amounts as low as 20 ng (2 × 10-6 mol), was injected into detached potato leaves. The differential activity of the peptides on hosts and non-hosts of the fungus, as well as on susceptible and tolerant cultivars, indicated host specific properties. An immunofluorescence assay detected the toxin in potato stems before extensive colonization occurred, suggesting that the toxin may be translocated in advance of the growing hyphae.
The complete amino acid sequence of alcohol dehydrogenase of Thermoanaerobium brockii (TBAD) is presented. The S-carboxymethylated protein was cleaved at methionine residues (with cyanogen bromide) to provide a set of 10 nonoverlapping fragments accounting for 90% of the sequence. These fragments were then overlapped and aligned, and the sequence was completed by using peptides generated by proteolytic cleavage at lysine residues (with Achromobacter protease I). The protein subunit contained 352 amino acid residues corresponding to a molecular weight of 37 652. The sequence showed about 35% identity with that of the prokaryotic Alcaligenes eutrophus alcohol dehydrogenase and about 25% identity with any one of the eukaryotic alcohol/polyol dehydrogenases known today. Of these, only 18 residues (5%) are strictly conserved:11 Gly, 2 Asp, and 1 each of Cys, His, Glu, Pro, and Val.
The effect of the thymic hormone, THF-γ2, on the immunocompetence of 5-fluorouracil (5-FU)-treated BALB/c mice, bearing MOPC-315 tumor, was examined. Treatment of noninoculated or tumor-bearing mice with THF-γ2 after 5-FU injection, resulted in an increase in the antibody response to sheep red blood cells and in the allogeneic response in spleen cell cultures and had no effect on the concanavalin-A-induced interleukin-2 secretion beyond that caused by 5-FU alone. Treatment with either 5-FU alone or 5-FU and THF-γ2 resulted in restoration to normal values of Lytl- and L3T4-positive populations in tumor-bearing mice. THF-γ2 prolonged the survival time of mice bearing MOPC-315 tumor beyond that observed in mice treated with 5-FU alone.
Thymic humoral factor ϒ2 (THF-ϒ2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-ϒ2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 μg of THF-ϒ2/1000 kg of thymus tissue. The concentration of THF-ϒ2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-ϒ2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-ϒ2 retains essentially all of the biological activity of the thymus extract from which it is derived.
cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma‐chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross‐hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H‐chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H‐chains, a mechanism which is not operative in the chicken L‐chain locus. The most notable among the chicken Igs is the so‐called 7S IgG because its H‐chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H‐chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8‐10 H‐chain genes.
The feasibility of using Thymic Humoral Factor (THF) for immunomodulation in asymptomatic male homosexuals was evaluated in a study on fifteen subjects with T-cell impairments, selected on the basis of a 2SD reduction in T helper/inducer (T4+) cells and one additional lymphocytic defect. Following two biweekly courses of treatment, mean relative increments of T4+ (P
Cytomegalovirus causes T cell immune impairment in infected mice, reflected by a decreased response to the T cell mitogens PHA and Con A, and reduction of Con A-induced IL-2 secretion. Concomitantly, a marked increase in the spleen weight of infected mice was found. Histological examination of the liver revealed focal hepatitis. These signs of infection lasted from day 5 to day 14 after infection when mice slowly started to recover. Systemic treatment of MCMV-infected mice with thymic humoral factor (THF) resulted in a reconstitution of the mitogenic responses, IL-2 secretion, normalization of spleen weight and recovery of liver inflammation. Unlike other thymic hormones, THF did not affect interferon synthesis and NK cytotoxicity in infected mice. It is concluded that THF restores immune competence of mice immunosuppressed as a result of MCMV infection, through modulation of the T cell compartment.
The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP‐Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.
Keywords: Immunology; Pharmacology & Pharmacy
Elastin was fully solubilized by digestion with elastase I or elastase II. Each digest was separated into high‐molecular weight and low‐molecular weight fractions that were characterized by the correspondence to their amino acid content, N‐terminal sequence and C‐terminal amino acids. It was found that although the relative amount of amino acids in the low‐molecular weight fraction obtained by digestion with elastase I was lower than in digestion with elastase II, no major difference in the type of bonds cleaved in the low‐ or high‐molecular weight fractions of each digest could be seen. There is, however, a remarkable difference in the type of bond cleaved by the two enzymes. While elastase I cleaves mostly Ala‐Ala and also Ala‐Gly bonds, elastase II hydrolyzes Leu‐Ala, Leu‐Gly, Phe‐Ala, Phe‐Gly and Tyr‐Ala, Tyr‐Gly bonds. Theoretical calculations led us to suggest both digests are composed of cross‐linked peptides that vary not only in the molecular size but also in the number of cross‐links found in peptides of the same size.
An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain α2-adrenergic receptors, has been isolated. The new compound, designed clonidine-displacing-substance (CDS), has been partially purified by ion exchange chromatography, zone electrophoresis and high performance liquid chromatography (HPLC). CDS binds specifically to α2-adrenergic receptors by competing with either α2-adrenergic agonists or α2-antagonists, but has no effect on the specific binding of [3H]prazosin to α1-adrenergic receptors in rat brain membranes. In the course of isolation, CDS was shown to be neither the endogenous neurotransmitter (-)norepinephrine (NE) nor the guanyl nucleotide GTP which lowers the specific binding of α2-agonists to the α2-adrenergic receptors.
To investigate the role of the leader peptide in modulating secretion from living cells, we injected a synthetic peptide into Xenopus oocytes. The peptide consisted of the NH2-terminal leader sequence of mouse immunoglobulin light chain precursor. We found that the leader peptide has two different roles in regulating secretion from the oocytes. First, it competitively inhibits the synthesis of secretory and membrane proteins but not of cytoplasmic proteins. The inhibition occurs both with oocyte proteins and with proteins directed by coinjected myeloma mRNA. The inhibition reaches a maximum 2 hr after injection and decays within 3 hr. It appears to be mediated through the cell membrane, because 125I-labeled leader peptide segregates into the membrane fraction of microinjected oocytes simultaneously with the interference with methionine incorporation. A second role of the microinjected leader peptide is to induce a rapid acceleration in the rate of export of secretory proteins from the oocyte. The maximal enhancement effect is obtained upon injection of 50 ng of leader peptide per oocyte. It is not merely due to the small size, negative charge, or hydrophobicity of the peptide, because enhanced secretion does not occur when glucagon, poly-L-glutamic acid, or Triton X-100 is injected. Furthermore, immunoreaction of the peptide with specific antibodies prior to microinjection prevents the accelerated export. Our observations indicate that in Xenopus oocytes, the leader peptide is involved in both translocation and later step(s) in the secretory pathway.
The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one κ species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two κ species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5′ of the κ coding region; the rearrangement site is within the κ cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two κ segments were mapped at 2 and 4.3 kb 5′ from the κ coding region. Therefore, the rat κ cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human jk clusters. In the region between κ and the expressed jk of IR52 myeloma DNA, an XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3′ from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat vk probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related κ genes.
The mRNA molecules coding for immunoglobulin κ chains of different subgroups were isolated from three rat myeloma tumors (IR102, IR487 and IR52). The mRNAs directed the cell-free synthesis of precursor molecules in which leader peptides (24-20 residues long) preceded the N-termini of the mature L-chains. All precursors had N-terminal initiator methionine residue. The complete primary structure of the IR102 leader, and the partial sequences of the IR487 and IR52 leaders were determined. The leader peptides differed extensively in sequence (> 37%), similarly to mouse Ig leader peptides of different subgroups. However, the rat leaders had the invariant residues Gly-4, Leu-9, Leu-10 and Met-20 (observed in mouse), that presumably are functionally significant and are conserved in evolution (Schechter et al., Fed. Proc. 1979, 38, 1839). We predict that in rat genome, as in mouse genome, the DNA coding for the leader and V-region are not contiguous, and that the invariant Gly-4 codon marks the 3′ end of the leader coding segment.
There is considerable evidence to support a common evolutionary origin for some of the polypeptide hormones of the anterior pituitary gland and those of the placenta. This is particularly true for the pituitary gonadotrophins (follicle-stimulating hormone and luteinizing hormone) and chorionic gonadotrophin, and for pituitary growth hormone (and to a lesser extent pituitary prolactin) and placental lactogen. These relationships are based principally on similarities in the covalent structure of these related hormones and on overlapping aspects of biological and immunological activity.'-6 In this manuscript, we will focus on placental lactogen and growth hormone, reviewing briefly some of the relationships of the mature hormones and discussing new studies related to their biosynthesis and the homology in structure of their precursor proteins.
Monolayers of granulosa cells from preovulatory follicles were incubated for 3 h with 125I-human chorionic gonadotropin (hCG) and subsequently for 5 h in the presence or absence of a saturating dose of unlabeled hormone at 37°C. Distribution of the labeled hormone during the first and second incubations was followed by electron microscopic autoradiography. After 2 h and 3 h of exposure to Y-hCG most of the label (80% and 69%, respectively) was associated with the cell membrane, whereas the remaining radioactivity was located within the cytoplasm. At the end of the second incubation most of the cell-bound label (73%) was internalized and found mainly in lysosome-like structures, whereas 30-40% of the total radioactivity was released into the medium. Further release of the bound radioactivity to the medium (up to 70-80%) was evident at the end of longer incubation periods (up to 24 h). Most of the released radioactivity was associated with mono- and diiodotyrosine as determined by ion exchange chromatography. During the first incubation, the cells were desensitized to hCG, as indicated by lack of response in 3’,5’-cyclic AMP accumulation to challenge with fresh hormone. The data suggest that desensitization precedes extensive internalization of the receptor-hormone complex and that the internalized hormone is transported to lysosomes in which degradation may take place.
The cell-free translation product of human placental lactogen mRNA is a precursor molecule larger than the mature hormone that circulates in plasma. To determine the structure of pre-placental lactogen, the poly(A)-rich RNA fraction of term placenta was isolated and translated in a wheat germ cell-free system. The mRNA programmed the synthesis of a major protein, 3000 daltons larger than placental lactogen, that was specifically precipitated by hormone antibodies. The immunoprecipitated protein was labeled separately with 20 radioactive amino acids and subjected to sequence analysis. The results showed the synthesis of pre-placental lactogen in which an extra piece 25 residues long preceded the NH2 terminus of the mature protein. The structure of the extra piece is as follows: Met-Pro-Gly-Ser-Arg-Thr-Ser-Leu-Leu-Leu-Ala-Phe-Ala-Leu-Leu-Cys-Leu-Pro-trp-Leu-Gln-Glu-Ala-Gly-Ala. Met1 is the initiator residue because only initiator [35S]Met-tRNA1(Met), but not internal [35S]Met-tRNA2(Met), donated NH2-terminal methionine. The structure of the extra piece showed little homology with that of unrelated hormones but striking homology (64%) with the extra piece of rat pre-growth hormone. Most amino acid substitutions involved a single base change in the codon. Mature human placental lactogen and rat growth hormone have 59% homology in sequence. Thus, our findings povide additional evidence to support the common evolutionary origin of these hormones, not only of the mature proteins but also of the extra piece segments.
Opsin, the apoprotein of the visual pigment rhodopsin, is synthesized on membranes of the rough endoplasmic reticulum and subsequently passes through the Golgi apparatus to the rod outer segment. This pathway parallels the early stages of biosynthesis of some secretory proteins and viral membrane glycoproteins. Most of these proteins are initially synthesized as precursor molecules with a short-lived hydrophobic extra peptide segment at the NH 2 terminus. Therefore we investigated whether or not the immediate translation product of opsin mRNA contains a similar short-lived NH 2-terminal extra peptide. The mRNA coding for opsin was isolated from bovine retina polysomes precipitated by antibodies to opsin. The mRNA directed the cell-free synthesis of a protein comparable in size to opsin that was specifically precipitated by anti-opsin antibodies. Sequence analyses of the immunoprecipitated protein labeled with six radioactive amino acids are reported. The partial sequence of the cell-free product corresponds exactly to the published NH 2-terminal segment of native opsin (21 residues long) and extends beyond this region. Met-1 was shown to be the initiator methionine residue. The finding essentially rules out the possibility that Met-1 was preceded by a peptide that was rapidly cleaved. Thus opsin, and not a precursor, is the immediate product of opsin mRNA translation.
The mRNAs of immunoglobulin (Ig) light (κ and λ) and heavy chains of mouse and rat direct the cell-free synthesis of precursors in which extra peptide segments (18-29 residues long) precede the N-termini of the mature proteins. The complete primary structures of the extra pieces of nine precursors were determined. The extra pieces exhibit sequence variability (up to 73%) that follows the pattern of heterogeneity of the V-regions; they are enriched with hydrophobic residues and all contain the initiator methionine. These features indicate: 1) the V-gene is larger than hitherto realized; 2) the extra pieces may function as a new recognition system; 3) the synthesis of Ig is contingent on the nucleotide sequence coding for the extra piece; 4) the role of the hydrophobic extra piece may be to favor interaction of the precursor with cell membranes such as the endoplasmic reticulum where it is cleaved to yield mature protein destined for secretion, and the plasma membrane to provide the antigen-recognizing receptor. Sequence analyses established mature H and L chains in the membrane-Ig of plasmacytoma and normal spleen cells. Frequency distribution analysis of amino acids (FDA) is a procedure developed to study the heterogeneous V-regions expressed in normal spleen. A recognition pattern for the enzyme(s) cleaving the extra piece is indicated from some residues (Gly, Leu) constant in all Ig extra pieces studied. The evolutionary divergence of L chains of different species (rat and mouse) and of different types (κ and λ) is reflected in the sequence homology of their extra pieces. Minor mRNA species (probably incompletely processed L chain HnRNA) direct the cell-free synthesis of a series of large proteins (mol wt up to 80.000) containing L chain peptides, suggesting the possibility of one gene coding for several related proteins. The precursor of the C(κ)-region fragment contains an extra piece similar to the extra piece linked to the V-regions. This indicates: 1) the DNA coding for the extra piece (Xp-DNA) is not directly linked to the DNA segment that encodes the mature (secreted) V-region; 2) independent expression of the C-gene is associated with translocation of the Xp-DNA to the C-gene.
The mRNA molecules coding for the MOPC‐41 immunoglobulin light chain direct the cell‐free synthesis of two precurosors in which 22 or 20 amino acid residues precede the NH2‐terminus of the mature light chain. The two NH2‐terminal residues of the long extra piece (22 residues) are missing in the shorter extra piece (20 residues), in the other 20 positions both extra pieces have an identical sequence. The sequences are: in the long extra piece, Met‐Asp‐Met‐Arg‐Ala‐…, in the short extra piece, Met‐Arg‐Ala‐… [Burstein, Y. and Schechter, I. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 716–720]. To study the relation between these molecules we analysed the amino acid sequence of precursors synthesized in the presence of the initiator [35S]Met‐tRNA1Met or internal [35S]Met‐tRNA2Met as sole source of label. The results show that the initiator [35S]Met‐tRNA1Met inserts [35S]methionine only at the NH2‐terminal position; the internal [35S]Met‐tRNA2Met does not label Met‐1 but inserts [35S]‐methionine inside the polypeptide chain at correct positions. These findings demonstrate that the NH2‐terminal methionine, present in both precurosors, is the initiator residue. Therefore, the two precursors are the direct product of mRNA translation, and the short precursor does not originate from the longer precursor by cleavage of the Met‐Asp dipeptide of the long extra piece. The identification of initiator methionine strengthens the contention that the precursor is synthesized intracellularly. Sequencing of precursors labelled with either [35S]Met‐tRNAMet species yielded discrete radioactive peaks of NH2‐terminal or internal methionines, without cross‐contamination. These results provide positive and controlled characterization of both the initiator and internal tRNAMet species, using a natural mRNA. The possibility of two initiation sites for translation on a single mRNA molecule is discussed.
The mRNA molecules coding for mouse immunoglobulin (Ig) light (L) chains program the cell-free synthesis of precursors in which extra peptide segments precede the N-termini of the mature proteins; i.e., the extra piece is linked to the variable (V) region (the amino terminal half of the L chain). The complete primary structures of the N-terminal extra pieces of three k-type and three X-type L-chain precursors were determined. Despite the fact that the extra pieces differ extensively in sequence (up to 73%) they share the following features: a high percentage of hydrophobic residues (69-75%), they are of comparable size (19-22 residues long) and contain fairly long peptide segments (16-19 residues) devoid of any charged residues, a few amino acids are frequently repeated (e.g., five leucines, five serines, and three isoleucines in RPC-20 extra piece), and methionine is the N-terminal residue. These structural features and other experiments indicate that (1) the V region is larger than hitherto realized; (2) the precursor seems to be the immediate translation product in the cell, since the N-terminal Met was identified as the initiator residue; (3) in the cell the precursor is short lived, and the maturation process (cleavage of the extra piece) may regulate secretion of the mature protein; (4) the physiological role of the hydrophobic extra piece may be to favor interaction of the precursor with cell membranes. Two targets for interaction are considered: the endoplasmic membranes and the cell-surface membrane. The N-terminal extra piece can undergo translocation from the V to the C region, as deduced from characterization of the precursor of the k-type constant (C) region polypeptide fragment (the carboxy terminal half of the L chain) which was found to contain an N-tenninal extra piece (17 residues long) with a primary structure identical to that of the extra piece linked to one of the V regions in whole L-chain precursors. These findings can be formally explained by the two genes-one Ig chain hypothesis, if we assume that the DN A coding for the extra piece (Xp DNA) is a constitutive part of the V gene. Alternatively, we raise the speculation that three genes may code for one Ig chain, where the Xp DNA represents a third distinct gene.
Plasma membranes were prepared from MOPC-321 mouse myeloma cells incubated with [3H]leucine. The L-chain from the purified plasma membranes was isolated, it was subjected to radioactive sequence analysis, and leucine was found at positions 4, 11 and 15. This sequence matches with that of the mature L-chain (Leu at positions 4, 11, 15, etc.) and differs from that of the L-chain precursor that contains a hydrophobic N-terminal extra piece (Leu at positions 6, 7, 8, 11, 12, 13, 24, 31, 35, etc.). This result establishes mature L-chain in the surface membrane of plasmacytoma cells.
A new porcine pancreatic serine protease, elastase II (Ardelt, W. (1974), Biochim. Biophys. Acta 341, 318-326), was further purified by removing the accompanying proteases on a turkey ovomucoid-Sepharose column. The purified enzyme was found to be homogeneous by ultracentrifugal sedimentation analysis (s20,w = 3.16), by electrophoresis on celllulose acetate and polyacrylamide gels at different pHs, by sodium dodecyl sulfate gel electrophoresis, and by electrofocusing in polyacrylamide gels. Elastase II is composed of about 250 amino acids, with a molecular weight of 26 500, and an NH2-terminal amino acid sequence which strongly resembles the B chain of bovine chymotrypsin B. Preliminary experiments indicated that it may be composed of two chains held together by a disulfide bond in which the NH2-terminal half-cystine participates. Elastase II has no activity towards specific substrates of porcine elastase I (EC 3.4.21.11) but exhibits characteristic chymotrypsin specificity. It has an extended binding site but like in other chymotrypsins the subsite S1 plays a dominant role in the binding. Similar to elastase I, elastase II solubilizes elastin by hydrolyzing about 6% of the peptide bonds. However, while elastase I hydrolyzes Ala-Ala and Ala-Gly peptide bonds, elastase II hydrolyzes the bonds formed between leucine, phenylalanine, or tyrosine with glycine or alanine. It seems, therefore, that the specificity of elastase II is completely different from that of elastase I. Elastase II also differs from porcine chymotrypsin A, B, and C, but strongly resembles a previously described proelastase B, which upon activation hydrolyzes elastin and exhibits chymotrypsin specificity.
Oxidation of methionine residues of chicken ovoinhibitor with N-chlorosuccinimide resulted in a selective loss of its inhibitory activities. While trypsin inhibiting activity was not affected at all, half of the chymotrypsin-inhibiting activity and all of the elastase inhibiting activity were lost. Electrophoretic and affinity chromatography studies indicated that the 50% loss of the chymotrypsin-inhibiting activity resulted from the inactivation of one of its two chymotrypsininhibiting sites rather than from a decrease in the binding constants of both sites. Oxidation of ovoinhibitor-chymotrypsin and ovoinhibitor-elastase complexes with excess of N-chlorosuccinimide indicated that the complex formation in each case protected the site that binds the enzyme which participated in the complex, but did not protect the site that binds the other enzyme. Quantitative estimation of the number of oxidized methionine residues in the ovoinhibitor isolated from the complexes has shown that in each complex about one methionine residue was protected from oxidation. Nitrophenylsulfenylation of the single tryptophan residue of ovoinhibitor did not affect its inhibitory activities at all.
The mRNA coding for the κ-type constant region (Cκ) was purified from two clones derived from the MPC-11 mouse myeloma. This mRNA directs the cell-free synthesis of a Cκ precursor (molecular weight, about 15,000) in which an extra piece, 17 residues long, precedes the NH2-terminal residue (Ala109) of the Cκ region. The partial sequence of the extra piece is: Met-X-Thr-Asp-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-X- (X is unknown). Met1 was shown to be the initiator methionine. The sequence of the Cκ extra piece is completely different from any known sequence preceding residue Ala109 in whole light (L) chains, thus establishing that the Cκ-region mRNA could not have originated from mRNA coding for the whole L chain. The structural features of the Cκ extra piece (marked hydrophobicity, size, and a methionine at the NH2-terminus) are identical to those characteristic of the NH2-terminal extra piece linked to the variable (V) region of whole L-chain precursors. In addition, the Cκ extra piece and the extra piece linked to the V region of MOPC-321 L chain have 70% sequence homology. These findings can be explained by the two genes-one Ig chain hypothesis, if we assume that the DNA coding for the extra piece (xp-DNA) is a constitutive part of the V gene. According to this model, the Cκ-region mRNA could have originated from: (i) translocation of this V gene to the C gene, deletion of the entire mature V gene, and “end-to-end” repair of the remaining xp-DNA to the C gene; (ii) translocation to the C gene only of the xp-DNA portion of the V gene. Alternatively, we may assume that the xp-DNA is not covalently linked to the mature V gene at all times, as might be the case for the DNA of hypervariable regions presumed to be in episomes. This raises the intriguing speculation that the xp-DNA represents a third distinct gene, designated xp-gene. The presumed xp-gene may be involved in the regulation of gene transcription: when linked to the mature V gene it initiates a chain of events leading to whole L-chain mRNA formation; when attached to the C gene it leads to its transcription to provide the C-region mRNA.
The mRNA molecules coding for three mouse immunoglobulin λ-type light (L) chains (MOPC-104E λ1, RPC-20 λ1, MOPC-315 λ2) program the cell free synthesis of precursors larger than the mature proteins. Radioactive amino acid sequence analyses of each of the three precursors labelled with [3H]alanine, [3H]serine, [3H]glutamine, [3H]glutamic acid and [3H]threonine showed that an extra piece, at least 18 residues long, is linked to the N terminus of the mature L chains. The N terminal extra peptide segment may be 19 residues long, since analyses of precursors labelled with [35S]methionine indicated an additional N terminal methionine residue which was recovered in low yields. Presumably this is the initiator methionine, which is known to be short lived in eukaryotes. The mature forms of MOPC 104E, RPC 20 and MOPC 315 λ L chains are blocked at the N termini by pyrrolid 2 one 5 carboxylic acid (pyroglutamic acid). Sequence analyses of precursors labelled with [3H]glutamine and [3H]glutamic acid showed incorporation only of glutamine in a position that matches with the position of pyrrolid 2 one 5 carboxylic acid in the mature forms of all three precursors, and incorporation of glutamic acid in other positions. The data showed the absence of glutamine glutamic acid interconversion, since the radioactive peaks obtained from either 3H labelled amino acid were discrete, and free from cross contamination. These results prove that glutamine is the precursor amino acid of pyrrolid 2 one 5 carboxylic acid at the N termini of the mature MOPC 104E λ1, RPC 20 λ1 and MOPC 315 λ2 L chains. Thus the formation of pyrrolid 2 one 5 carboxylic acid by cyclization of glutamine is a post translational event which occurs after, or concomitant with, cleavage of the extra piece from the precursor to yield the mature L chain. The variable (V) regions (110 amino acid residues) of mouse λ L chains are quite similar: when compared with that of MOPC 104E λ1 chain, the V region of RPC 20 λ1 chain differs in one residue, and the V region of MOPC 315 λ2 chain differs in 11 residues. The partial sequence data show that the N terminal extra pieces of the two λ1 L chain precursors have, so far, identical partial sequences; the extra piece of the λ2 L chain precursor differs from these in at least 3 out of 19 positions.
Tryptophanyl peptide bonds are selectively cleaved by N-chlorosuccinimide (NCS) under acidic conditions. All other peptide bonds are resistant to cleavage by this reagent. Optimal conditions for cleavage are: 2 equiv of NCS, pH 4–5, or 50–80% acetic acid for 30 min at room temperature. Under these conditions methionine residues are oxidized to methionine sulfoxides and cysteine to cystine. Other amino acids are not modified. The cleavage reaction was studied with several peptides containing tryptophan residues and yields of cleavage were in the range of 35–45%. The method was successfully applied to several proteins. In α-lactalbumin, Kunitz trypsin inhibitor, and apomyoglobin, selective cleavage of the expected tryptophanyl peptide bonds was obtained in 19–58% yield. The glucagon molecule was fragmented into two peptides in 32% yield.
The proteins programmed in the wheat germ cell free system by the mRNA coding for the MOPC 321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5 3H]leucine or [3 3H]serine, and were subjected to amino acid sequence analyses. Over 95% of the total cell free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L chain protein. In the precursor, 20 amino acid residues precede the N terminus of the mature protein. This extra piece contains one methionine residue at the N terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra piece moiety is quite hydrophobic. The extra piece of the MOPC 321 L chain precursor synthesized with the aid of the Krebs II ascites cell free system is of identical size and it has the same leucine sequence. This indicates that cell free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labeled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000000 fold) of unrelated non radioactive proteins.
mRNAs coding for mouse immunoglobulin light chains direct the cell-free synthesis of precursors in which extra peptide segments precede the NH2-termini of the mature proteins. The abundance (18-30%) of leucine residues in the extra piece indicates that it is quite hydrophobic [Schechter and Burstein (1976) Biochem. Biophys, Res. Commun. 68, 489]. Accordingly, we have determined the positions of all hydrophobic residues by sequencing two k-type light (L)-chain precursors that were labeled with: [3H]Ala, [3H]Val, [3H]Leu, [3H]Ile, [3H]Thr, [3H]Pro, [3H]Phe, [3H]Tyr, [3H]Trp, [35S]Met, and [35S]Cys. The partial sequences (and sizes) of the extra pieces obtained are: in MOPC-321 precursor, Met-X-Thr-X-Thr-Leu-Leu-Leu-Trp-Val-Leu-Leu-Leu-Trp-Val-Pro-X-X-Thr-X-(20 residues; X is unknown); in MOPC-41 precursor, Met-X-Met-X-Ala-Pro-Ala-X-Ile-Phe-X-Phe-Leu-Leu-Leu-Leu-Phe-Pro-X-Thr-X-Cys- (22 residues). Despite the fact that these extra pieces differ extensively in sequence (68%), both of them are highly enriched with hydrophobic residues (75% in MOPC-321, 73% in MOPC-41). This marked hydrophobicity suggests that the extra piece favors interaction of the precursor with cell membranes, in a manner similar to the function of the "hydrophobic domain" of membrane-bound proteins (e.g., glycophorin). We propse that the hydrophobic extra piece directs most precursor molecules to the endoplasmic reticulum, where they are cleaved to yield mature L chain destined for scretion; a few precursor molecules escape cleavage and are embedded in the cell surface to serve as the antigen-recognizing receptor. The probability that the Leu-Leu-Leu-Trp-Val sequence occurs by change is 1.6 X 10(-8). Therefore, the data provide evidnece for duplication of a short DNA segment in the structural gene coding for the MOPC-321 precurosr. Duplication with inversion is also indicated from inverted repetition of the Phe-Lue-Leu sequence in the extra piece of the MPOC-41 precursor.
The proteins programmed in the wheat germ cell-free system by the M-41 L-chain mRNA were labeled with [35S]Met, [3H]Leu, [3H]Ile or [3H]Pro, and were subjected to amino acid sequence analyses. The results showed that this mRNA directs the synthesis of two precursors in which 22 or 20 residues precede the N-terminus of the mature M-41 L-chain. Partial sequence of the extra pieces in these precursors are: {A figure is presented} Considering the role of Met in the initiation of protein synthesis in eukaryotes, that both precursors contain N-terminal Met, the proximity of the two methionines, and the complete sequence homology, it is reasonable to assume that the two precursors originated from the initiation of M-41 mRNA translation at two points (Met-1 and Met-3 codons). The extra piece in the precursors is coupled to the variable(V)-region of the L-chains. The precursors of M-41 and M-321 L-chains (which are of different subgroups) contain extra pieces which differ in size and sequence. These findings imply that the extra piece is part of the V-region, i.e., that the gene coding for the immunoglobulin V-region may be larger than hitherto known.
To gain information on the origin of antibody diversity (somatic mutation or germ line hypothesis) it is necessary to determine the number of V region genes. For this purpose the capacity of a distinct V region probe to hybridize and quantify V genes of the same and different subgroups should be established. Relevant information on this issue was obtained from the extent of cross hybridization of a distinct L chain cDNA with mRNAs coding for L chains of the same and different subgroups. The results indicated that: V regions of similar amino acid sequence are coded by similar nucleotide sequence (this is not self evident because of the degeneracy of the genetic code); the nucleic acid probe to one V region may anneal and quantify V genes of members of the same subgroup. Molecular hybridizations of the cDNA probe with nuclear DNA showed that: the number of kappa type C genes is small (about 2 per haploid genome); the number of V genes presumably is also small; there is no amplification of these genes in myeloma cells that produce large amounts of the Ig. These results support the somatic mutation model for the generation of antibody diversity. New information on the structure and controlled expression of Ig genes was obtained from the study of L chain precursors, which are the immediate product of L chain mRNA translation in vitro. In the precursors extra peptide segments (19 to 22 residues in length) precede the N terminus of the mature L chain. Amino acid sequence analyses of the precursors provide evidence that: the gene coding for the V region is larger than hitherto known; duplication of a short DNA segment occurred in the structural gene coding for the MOPC 321 precursor; translation of the L chain mRNA may be contingent on the nucleotide sequence coding for the extra piece; cleavage of the extra piece may regulate secretion of mature L chain; the extra piece is remarkably hydrophobic, suggesting that the role of the extra piece is to anchor the precursor in cell membranes, in a manner similar to the function of the hydrophobic domain of membrane bound proteins. The authors propose that most precursor molecules are directed to the endoplasmic reticulum where the extra piece is cleaved to yield mature Ig destined for secretion; a few precursor molecules escape cleavage and are anchored by means of the hydrophobic extra piece in the cell surface membrane to serve as the antigen recognizing receptor.
Analyses of amino acid sequences of the total cell free products programmed by the mRNA of MOPC 104E λ light (L) chain show that over 95% of the products have sequences of a distinct protein that correspond to the L chain precursor. In this precursor an extra piece is coupled to the NH2 terminus of the mature L chain. Analyses of products labeled with [3H]alanine, [3H]leucine, and [3H]proline demonstrate that the extra piece is composed of at least 18 residues. Analyses of [35S]methione labeled product indicate that the extra piece may contain an additional NH2 terminal methionine, which is detected in about 10% of the molecules. Partial recovery of the NH2 terminal methionine (alanine, leucine, and proline are recovered in yields close to theoretical,>95%) suggests that it is the initiator methionine, which is known to be short lived in eukaryotes due to rapid hydrolysis. Thus, the extra piece seems to be 19 residues in length, and it contains one methionine at the NH2 terminus, three alanines at positions 2, 12, and 17, and five leucines at positions 6,8,10,11, and 13. The close gathering of leucine residues, as well as their abundance(26%), suggest that the extra piece would be quite hydrophobic. Hydrophobicity seems to be a general property of the extra piece, since similar clusters of leucine were found in the precursors of 3(kappa) L chains [Burstein, Y. & Schechter, I. (1976) Biochem. J. 157,145-151]. The NH2 terminus of the mature MOPC 104Eλ L chain is blocked by pyrogultamic acid. The fact that in the precursor a peptide segment precedes this NH2 terminus establishes that pyroglutamic acid is not the initiator residue for synthesis of the L chain. Apparently, the pyroglutamic acid is formed by cyclization of glutamic acid or glutamine during cleavage of the extra piece to yield the mature L chain.
Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).
A comparison of the partial amino acid sequence of neutral protease A from B. subtilis with the structure of thermolysin (EC 3.4.24.4) from B. thermoproteolyticus reveals that these two proteins are homologous. Of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include 9 of the 13 residues previously identified as components of the active site of thermolysin. This similarity provides support for the hypothesis that the two enzymes have similar three dimensional structures and a common mechanism of action. Since these enzymes differ markedly in their resistance to heat inactivation, a comparison of their structures may eventually provide a chemical basis for explaining the differences in their thermal stability.
Thermolysin from Bacillus thermoproteolyticus and the neutral proteases of Bacillus subtilis are neutral metalloendopeptidases having similar size, metal content, amino acid composition, and substrate specificity. Recent evidence indicates that their amino acid sequences have regions of identity, suggesting that these enzymes have diverged from a common ancestor. Problems of self-digestion, which have been encountered in the purification of these and other proteolytic enzymes, can be minimized by rapid procedures of affinity chromatography. Because Cbz-Gly-D-Phe is a competitive inhibitor of both enzymes, affinity adsorbents were developed using Gly-D-Phe covalently attached by appropriate spacers to a Sepharose matrix. The enzymes were selectively adsorbed at low pH and eluted by raising the pH. Three effective adsorbents have been described that can be used interchangeably for thermolysin and for neutral protease and differ only in the length of their spacers. Preparation of the simplest adsorbent is described in this chapter.
Tryptophan residues of bovine α-lactalbumin, one of the two protein components of lactose synthetase, were selectively sulfenylated with 2-nitrophenylsulfenyl chloride. When the reaction was performed in 0.1 M acetic acid for 1 hour, a modified protein containing 1.0 nitrophenylsulfenyl tryptophan residue per α-lactalbumin molecule was formed and isolated in 42% yield. Upon reduction, carboxymethylation, and trypsin digestion, this sulfenylated α-lactalbumin (NPS1-αLA) yielded two yellow peptides. Amino acid composition of these peptides indicated that in NPS1-αLA, tryptophan residues 60 and 118 were equally sulfenylated to the extent of 0.5 nitrophenylsulfenyl tryptophan per α-lactalbumin. NPS1-αLA cross-reacts with anti-α-lactalbumin antibodies, but, unlike the native protein, it has no biological activity in the lactose synthesis reaction, and its electrophoretic mobility on polyacrylamide gels is different from that of native α-lactalbumin.
Thermolysin is inhibited by ethoxyformic anhydride in the pH range of pH 6-8. The inhibition is reversed by hydroxylamine and prevented by the competitive inhibitor Cbz-L-phenylalanine. Although several ethoxyformyl groups are introduced into the enzyme molecule, inactivation can be correlated with modification by a single group. Spectral analysis indicates that the removal of an ethoxyformyl group from one histidine residue per molecule is responsible for the reactivation of the enzyme.
Affinity chromatographic systems are described for the purification of neutral metalloendopeptidases on columns of acetyl-D-phenylalanine or succinyl-D-leucine covalently linked to Sepharose by spacers of various lengths. The neutral proteases of B. subtilis are separated in a single chromatographic procedure from all other proteins of the culture filtrates and subfractionated into two active species. An analogous chromatographic system is effective in the purification of thermolysin of B. thermoproteolyticus.
Bovine a-lactalbumin was reduced by dithioerythritol in aqueous solutions at pH 7,0 in two steps: a fast step followed by a much slower one. At the end of the fast step the partially reduced protein was carboxymethylated with 2-iodoacetic acid, and a product containing two carboxymethylcysteine residues per a-lactalbumin (2CM-aLA) was isolated in about 95 % yield. By fragmentation of 2CM-aLA and characterization of the fragments, it has been established unequivocally that cystine I-VIII was the only reduced and carboxymethylated disulfide bond in 2CM-aLA. No other residues have been effected. 2CM-aLA is a homogeneous product, as was judged from its electrophoretic mobility on ion-exchange columns and on polyacrylamide disc electrophoresis in several different pH systems. Although the ability of 2CM-aLA to bind anti-a-lactalbumin antibodies cannot be distinguished from that of the native protein, 2CM-aLA has only about 50% of the biological activity of a-lactalbumin in the lactose synthesis reaction, and about 35% of the inhibitory capacity of a-lactalbumin in the inhibition of W-acetyllactosamine synthesis.
A method of possible general utility for the location of bufied and exposed tyrosine residues in proteins has been developed using bovine pancreatic ribonuclease as a model. Exposed tyrosine residues were acetylated with N-acetylimidazole or acetic anhydride and were thus protected from cleavage by N-bromosuccinimide. The partially acetylated protein was reduced with dithioerythritol, S-carboxy-methylated with 2-iodoacetic acid, and subjected to cleavage with N-bromosuccinimide. By determining the bonds that could be cleaved with N-bromosuccinimide, tyrosine residues that were buried in the native molecule could be identified. The method was applied to partially acetylated ribonuclease. N-Bromosuccinimide cleavage and end-group analysis of the cleaved peptide bonds revealed that tyrosine residues 25, 92, and 97 were the only ones to be cleaved, and it was thus concluded that the other three tyrosine residues, namely, residues 73, 76, and 115, had been acetylated and therefore not cleaved by N-bromosuccinimide.
A single tryptophan residue in hen egg-white lysozyme was selectively sulfenylated by 2-nitrophenylsulfenyl chloride in aqueous solution at pH 3.5. The monosulfenylated lysozyme derivative (1-NPS-lysozyme) was purified by gelfiltration and by ion-exchange chromatography. Upon reduction, carboxymethylation, and trypsin digestion, 1 NPSlysozyme yielded only one yellow peptide. Amino acid composition and sequence analysis of this peptide showed that tryptophan-62 was the only sulfenylated residue in 1-NPSlysozyme. 1-NPS-lysozyme has the same electrophoretic mobility as that of the native enzyme, and cross-reacts with antilysozyme antibodies, but unlike the native enzyme it has no lytic activity on dead cells of Micrococcus lysodeikticus, and two of its peptide bonds are susceptible to digestion by trypsin.
Bovine pancreatic ribonuclease was found to react with the sulfhydryl reagents dithioerythritol or dithiothreitol in two steps: a fast step followed by a much slower one. The product at the end of the fast step was carboxymethylated and digested by pepsin, and the digest was analyzed by paper electrophoresis and chromatography. Only cystine bond IV-V was reduced during the fast reduction step. The partially reduced protein obtained at the end of the fast step reacts with either 1 or 2 moles of mercuric ions, yielding derivatives of the type -S-Hg-S- or -SHg+, respectively. The monomercury derivative was crystallized in a form which is very nearly isomorphous with the monoclinic crystalline form of native ribonuclease. The mono- and dimercury derivatives, as well as the carboxymethyl derivative of ribonuclease reduced at cystine IV-V, are enzymically as active as the native protein and are resistant to digestion by trypsin. Similarly, the mercury derivatives exhibit an optical rotatory dispersion which is identical with that of native ribonuclease. Three of their tyrosine side chains titrate abnormally as in native ribonuclease. The cystine bridge IV-V in bovine pancreatic ribonuclease thus seems to be completely unnecessary for enzymic activity or for maintaining the native macromolecular conformation of ribonuclease.
The NCAs of the N‐benzyl derivatives of β‐alanine, β‐DL‐aminobutyric acid, and β‐DL‐aminoisobutyric acid (nonplanar six‐membered rings) were prepared by reacting the corresponding N‐chloroformyl derivatives, obtained on reaction of the N‐benzyl amino acids with phosgene, with triethylamine. Contrary to the others, the NCA of N‐benzyl‐β‐alanine polymerized readily on heating in vacuo or in solution, using n‐hexylamine or methanolic sodium methoxide as initiators. With n‐hexylamine the molecular weights of the polymers obtained in benzene, dioxan, and dimethylsulfoxide, were in accordance with DP = [NCA]/[Initiator], as was found with conventional five‐membered ring NCAs of α‐amino acids. With sodium methoxide initiation, DPs of the polymers obtained were smaller than the (NCA)/[Initiator] ratio, contrary to what was found previously with α‐amino acid NCAs. The possibility that stereochemical factors are responsible for the differences in polymerization activity of various. N‐alkyl β as well as α amino acid NCAs is discussed.