Yigal Burstein

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.

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.

Isolation of an endogenous clonidine-displacing substance from rat brain

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.

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

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 ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol.

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.

Biological and immunochemical characterization of a low molecular weight phytotoxin isolated from a protein—lipopolysaccharide complex produced by a potato isolate of Verticillium dahliae Kleb

Abstract A low molecular weight (less than 3000) phytotoxic polypeptide fraction was partially purified from the protein-lipopolysaccharide (PLP) complexes produced in culture by a strain of Verticillium dahliae pathogenic on potato. The toxic fraction was host-specific and antigenically identical to the high molecular weight PLP. Immunodiffusion and immunofluorescence assays showed that a substance is present in stems and tubers of infected potato plants which was antigenically related to the low molecular weight toxin. When used as a tool for screening new potato varieties for tolerance to Verticillium wilt disease, the low molecular weight toxin was more reliable than the PLP because non-specific sensitivity to macromolecules was not observed.

Affinity chromatography of thermolysin and of neutral proteases from B. subtilis

Abstract 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 .

A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase

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: ΔT  1/260 min = + 8°C (temperature of 50% inactivation after incubation for 60 min), ΔT  1/2CD = +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. Proteins 2007.

Restrictin-P/Stromal Activin A, Kills its Target Cells Via an Apoptotic Mechanism

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.

[50] Thermolysin and other neutral metalloendopeptidases

Publisher Summary 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.

Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.

cDNA libraries of chicken spleen and Harder gland (a gland enriched with immunocytes) constructed in pBR322 were screened by differential hybridization and by mRNA hybrid‐selected translation. Eleven L‐chain cDNA clones were identified from which VL probes were prepared and each was annealed with kidney DNA restriction digests. All VL probes revealed the same set of bands, corresponding to about 15 germline VL genes of one subgroup. The nucleotide sequences of six VL clones showed greater than or equal to 85% homology, and the predicted amino acid sequences were identical or nearly identical to the major N‐terminal sequence of L‐chains in chicken serum. These findings, and the fact that the VL clones were randomly selected from normal lymphoid tissues, strongly indicate that the bulk of chicken L‐chains is encoded by a few germline VL genes, probably much less than 15 since many of the VL genes are known to be pseudogenes. Therefore, it is likely that somatic mechanisms operating prior to specific triggering by antigen play a major role in the generation of antibody diversity in chicken. Analysis of the constant region locus (sequencing of CL gene and cDNAs) demonstrate a single CL isotype and suggest the presence of CL allotypes.