Interaction of myotoxin a with artificial membranes: Raman spectroscopic investigation

Myotoxin a from the venom of Crotalus viridis viridis (prairie rattlesnake) is a small protein which is responsible for myonecrosis. It is a basic protein with 42 amino acid residues of known sequence. Three disulfide bonds give it a highly compact structure. Microscopic examination of the toxin's effects reveals that the most pronounced and earliest visible damage occurs intracellularly, in the sarcoplasmic reticulum membrane system of skeletal muscle. A better understanding of its mechanism of action is therefore of particular interest. The interaction of myotoxin a with artificial membranes (multibilamellar phospholipid dispersions) was investigated by using dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). Two regions of the Raman spectrum were examined for information: the C-H stretching region between 2800 and 3000 cm-1 and the C-C stretching region between 1000 and 1300 cm-1. The effects of myotoxin a on the thermotropic phase behavior of the artificial membranes were determined. This was done by monitoring three structurally sensitive Raman intensity ratios, I2932/2880, I2880/2850, and I1088/1126. It was found that myotoxin alpha destabilized the ordered structure of the gel phase of phospholipid bilayers. This effect was seen with both DMPC and DMPS. The pretransition of DMPC was perturbed by myotoxin a, while the main gel to liquid-crystal phase transition temperature was decreased. The effect of myotoxin a on the phase behavior of DMPS was found to be pH dependent with the least effect observed at low pH values. These results suggest the involvement of negatively charged phosphate groups of phospholipids in the interaction of myotoxin a with artificial membranes.     

Specificity of hemorrhagic proteinase from Crotalus atrox (western diamondback rattlesnake) venom

Hemorrhagic proteinase, HTb, isolated from Crotalus atrox (western diamondback rattlesnake) venom was studied for its specificity. HTb showed fibrinogenase activity, hydrolyzing the A alpha chain of fibrinogen first, followed by the cleavage of the B beta chain. HTb is different from thrombin and did not produce a fibrin clot. The degradation products of fibrinogen were found to be different, indicating that the cleavage sites in the A alpha and B beta chains are different from those of thrombin. N-Benzoyl-Phe-Val-Arg-p-nitroanilide was not hydrolyzed by HTb, although this substrate was hydrolyzed by thrombin and reptilase.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

Enzymes of vitamin B6 degradation. Purification and properties of two N-acetylamidohydrolases

alpha-(N-Acetylaminomethylene)succinic acid hydrolase (Compound A hydrolase, EC 3.5.1-) and alpha-hydroxymethyl-alpha'-(N-acetylaminomethylene)succinic acid hydrolase (Compound B hydrolase, EC 3.5.1-) were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. The two inducible enzymes catalyze Reactions 1 and 2, respectively, which release the first generally useful anabolic intermediates during growth of these organisms with (formula; see text) pyridoxine as a sole source of carbon and nitrogen. Compound A hydrolase is a monomeric protein of Mr 32,500 with aspartic acid as its NH2-terminal residue. Compound B hydrolase (Mr congruent to 205,000) is a multimer containing probably six identical subunits with glycine as the NH2 terminus. The two enzymes have quite different amino acid analyses, although both are high in Asx and Glx, lack tryptophan, and show similar stabilities to pH and temperature. Compound A hydrolase has a pI of 4.4, a Km of 3.3 microM, and a Vmax of 3.1 mumol X min-1 X mg-1 at pH 6.5 and 25 degrees C; no analogue substrates were found. Compound B hydrolase has a pI of 4.2, a Km of 25 microM, and a Vmax of 3.8 mumol X min-1 X mg-1 at 25 degrees C and pH 7.0; it also hydrolyzes Compound A slowly. Both enzymes are inhibited competitively by di- and tricarboxylic acids, itaconic acid being among the most effective. Sulfite inhibits both enzymes by a time-dependent mechanism not yet understood. The two amidases appear to differ greatly in architecture despite the similarity in properties and in the overall reactions they catalyze.     

蝮蛇毒抗凝血活酶组份及几种蛇毒粗毒对人红细胞和血小板的作用

蝮蛇毒抗凝血活酶组分及蝗蛇毒、圆斑蝰蛇毒和眼镜蛇毒粗毒,不仅能够水解血浆中的磷脂,而且还能水解完整人红细胞膜和完整人血小板膜上的磷脂。但是五步蛇毒和金环蛇毒粗毒却不能水解完整人血小板膜上的磷脂。 扫描电镜观察表明,由于抗凝血活酶组份和几种蛇毒粗毒的作用,人红细胞和人血小板的形态发生了巨大的变化;人红细胞由正常的双圆盘形变成带刺的小球,人血小板的外形变成蜂窝状。     

Novel extracellular enzymes (ligninases) of Phanerochaete chrysosporium

Abstract 3 New spectrophotometric enzyme assays were developed for the study of microbial lignin-degrading enzymes. The conversion of 2-methoxy-3-phenylbenzoic acid to 2-hydroxy-3-phenylbenzoic acid led to the discovery of an extracellular, aromatic methyl ether demethylase produced by the white-rot fungus Phanerochaete chrysosporium . The conversion of methyl 2-hydroxy-3-phenylbenzoate to 2-hydroxy-3-phenylbenzoic acid allowed the identification of an extracellular, aromatic methyl ester esterase produced by this fungus. The Phanerochaete sp. also excreted an enzyme complex that oxidized 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, probably to aliphatic products. All 3 novel enzyme activities were produced together with, and probably comprise a part of, the Phanerochaete ligninolytic enzyme complex. Unlike previously known ligninases, these enzymes did not oxidize 3,4-dimethoxybenzyl alcohol. All 3 were H₂O₂-dependent and were activated by Mn²⁺ ions.     

The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone

We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.