Tomoji Suzuki

Isolation and characterization of two phospholipase as from the venom of Agkistrodon halys blomhoffii

Abstract Two phospholipase As (phosphatide acyl-hydrolase, EC 3.1.1.4) found in the venom of Agkistrodon halys blomhoffii were purified by successive chromatographies on ion exchangers and gel filtration on Sephadex G-100. The purified enzymes, A-I and A-II, each gave a single band on disk electrophoresis on polyacrylamide gel and a symmetrical schlieren sedimentation pattern. On isoelectric focusing analysis each protein also gave a single protein peak with phospholipase activity. Enzyme A-I had a sedimentation constant of 1.8 S and an average molecular weight, determined by two different methods, of 13 800 (± 500). The protein was strongly basic, as indicated by its isoelectric point of pH 10.0. Enzyme A-II had a sedimentation constant of 1.9 S, a diffusion constant of 13.35·10 −7 cm 2 ·sec −1 , and an average molecular weight, determined by three different methods, of 13 700 (± 500). In contrast to enzyme A-I, enzyme A-II was acidic, as indicated by its isoelectric point of pH 4.0. Thus, the charge distributions of the protein molecules of the two enzymes differed significantly. Moreover, studies of circular dichroism in the spectral region between 210 and 310 nm revealed conformational differences between the side-chain chromophores of the two proteins, probably due to the tyrosyl and tryptophyl residues in these phospholipase molecules. Chemical analysis of the proteins of A-I and A-II gave, respectively, 14.44 and 15.45% total nitrogen and 2.98 and 3.18% total sulfur. Thus, both proteins had relatively high sulfur contents. However, no free sulfhydryl groups were detected on titration with p -chloromercuribenzoate (PCMB). Neither protein contained appreciable hexose, hexosamine or sialic acid.

Proteinase, arginineester hydrolase and a kinin releasing enzyme in snake venoms☆

Abstract The distributions of proteinase, arginineester hydrolase and kinin releasing enzyme in 32 commercially available snake venoms were studied. The endopeptidase activity in Crotalidae venoms was considerably higher than that in Elapidae venoms, but significant differences in potency were observed even between different samples of the same species. The crude venoms of the families Crotalidae and Viperidae had arginineester hydrolytic activity independent of proteinase activity. There are significant differences of the arginineester hydrolytic activities even in the venoms of different species of the Crotalidae and Viperidae, and the venoms of Elapidae had no esterolytic activity. The kinin releasing activity like arginine ester hydrolytic activity was greatest in venoms of Crotalidae. However, some venoms of Crotalidae, such as the venom of Agkistrodon actus and the venom of Trimeresurus flavoviridis showed slight kinin releasing activity.

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185: I. Preparation of partially purified enzyme system and its properties☆

Abstract 1. A tyrocidine-synthesizing system was partially purified from the crude extract of Bacillus brevis by ammonium sulfate fractionation, protamine sulfate treatment followed by another ammonium sulfate fractionation to a specific activity of about 20 mμmoles of tyrocidine formation per h per mg of protein. The partially purified system absolutely required Mg 2+ (or Mn 2+ ), ATP and the tyrocidine constituent amino acids for its activity. The optimum conditions for tyrocidine synthesis were found to be 2 mM Mg 2+ (or Mn 2+ ), 4 mM ATP, and pH 7.8 in the presence of 0.2–0.4 mM constituent amino acids. 2. AMP was formed from ATP in the process of tyrocidine synthesis and 1 molecule of ATP was required for the formation of one peptide bond. 3. The partially purified system catalyzed ATP-PP i exchange reactions in the presence of glycine, l -alanine, l -lysine and l -isoleucine, besides 9 tyrocidine constituent l -amino acids. The other non-constituent amino acids of tyrocidine did not promote the exchange reaction. 4. Column chromatography on DEAE-cellulose separated the partially purified system into two distinct components, Components I and II, both of which were required for tyrocidine synthesis.

Bradykinin-potentiating peptides from the venom ofAgkistrodon halys blomhqffii

Aus dem Gift vonAgkistrodon halys blomhoffii konnten durch Säulenchromatographie an Sephadex G-100, G-25 und CM-Sephadex C-50 fünf Peptidkomponenten isoliert werden, welche die kontrahierende Wirkung von Bradykinin auf den isolierten Meerschweinchendarm signifikant erhöhen.

Amino acid composition and end group analysis of cobrotoxin

Abstract Cobrotoxin, the crystalline toxin obtained from Formosan cobra venom, is composed of 15 common amino acids and is devoid of alanine, methionine and phenylalanine. The minimal molecular weight calculated from amino acid composition (62 amino acid residues) is 6949. The toxin consists of a single peptide chain cross-linked intramolecularly by four disulfide bonds. Leucine is in the amino-terminal position and asparagine in the carboxyterminal position.

The isolation and amino acid sequences of new pyroglutamylpeptides from snake venoms.

Die Strukturaufklärung von zwei neuen tryptophanhaltigen Peptiden im Schlangengift vonAgkistrodon halys blomhoffii ergeben:l-Pyroglutamyl-l-Glutaminyl-l-Tryptophan undl-Pyroglutamyl-l-Asparaginyl-l-Tryptophan. Diese Peptide sind in Schlangengiften der Viperidae- und Crotalidae-Arten verbreitet.

Structure of bradykinin-potentiating peptide containing tryptophan from the venom ofAgkistrodon halys blomhoffii

Es wird über die Strukturaufklärung eines Bradykinin-potenzierenden Peptids aus dem Gift vonAgkistrodon halys blomhoffii berichtet.

Snake venom NAD nucleosidase: its occurrence in the venoms from the genus Agkistrodon and purification and properties of the enzyme from the venom of A. halys blomhoffii.

Abstract Nicotinamide adenine dinucleotide nucleosidase (NAD glycohydrolase, EC 3.2.2.5) was demonstrated in venoms of various snakes. Among the venoms from 37 species of Viperidae, Crotalidae and Elapidae, venom of snakes in the genera Bungarus and Agkistrodon showed the highest activities. No NAD nucleosidase activity was detected in venoms of the Naja genus. A NAD nucleosidase found in the venom of Agkistrodon halys blomhoffii was purified by successive column chromatographies on DEAE-cellulose and DEAE-Sephadex A-50 and gel-filtration on Sepharose 6B. Final purification was over 25-fold with 20 per cent yield. The purified enzyme showed maximal activity at pH 7.5 and hydrolysed β-NAD+, NADP+ and 3-acetylpyridine adenine dinucleotide. No hydrolysis could be observed on α-NAD+, NADH, NADPH and NMN+. The Km values for β-NAD+, NADP+ and 3-acetylpyridine adenine dinucleotide were 8.3 × 10−4 M and 7.4 × 10−4 M, respectively. Over 60 per cent inhibition of β-NAD+ hydrolysis was observed in the presence of 10−3 M of HgCl2 and cysteine.

Distribution of proteinase inhibitors in snake venoms

Abstract Potent proteinase inhibitors, which inactivate bovine pancreatic trypsin, α-chymotrypsin, bovine plasma kallikrein and plasmin, were found in several snake venoms. The content of the inhibitor was highest in Vipera russelli venom. Proteinase inhibitors of the same type were demonstrated also in the venoms of five snakes of the Elapidae family; Hemachatus haemachatus (Ringhals cobra), Dendroaspis angusticeps (Green mamba), Dendroaspis polylepis (Black mamba), Naja nivea (Cape cobra) and Naja haje (Egyptian cobra). No proteinase inhibitors were demonstrated in several Crotalidae and Hydrophiidae venoms. Toxic polypeptides such as α-bungarotoxin, cytotoxin I and II ( Naja naja venom) and cardiotoxin ( Naja naja atra venom) did not show any inhibitory action on typical mammalian proteinases including trypsin, α-chymotrypsin, plasmin and kallikrein.

Primary structure of proteinase inhibitor II isolated from the venom of Russell's viper (Vipera Russelli)

Basic proteinase inhibitors of trypsin and related enzymes are widely distributed in the animal tissues including pancreas, lungs, parotide glands and liver [ 1 ]. A number of these have been purified and characterized, including their primary structures [2]. The use of these inhibitors as models for the study of structurefunction relationships is particularly attractive. Previously, two novel inhibitors which inhibit kallikrein, plasmin, c~-chymotrypsin and trypsin have been found in the venom of Russells viper, and one of them designated as RVV inhibitor II was characterized to be a polypeptide consisting of 60 amino acid residues [3]. This paper describes the complete amino acid sequence and locations of disulfide bridges in the inhibitor.