Sadaaki Iwanaga

BOVINE PLASMA COLD-INSOLUBLE GLOBULIN: GROSS STRUCTURE AND FUNCTION*

Bovine plasma CIg, like human CIg, is a glycoprotein with a molecular weight of approximately 450,000 daltons and consists of two homologous subunits, the alpha and beta chains. These subunits are covalently linked through disulfide bridges in their carboxyl terminal domains. The carboxyl terminal regions are presumed to contain the fibrin-reactive transamidation site. The covalent incorporation of CIg into fibrin has been conclusively demonstrated by isolation of the S-carboxymethyl derivative of the CIg-fibrin-alpha chain complex and by determination of its terminal amino acid sequences. Cold-insoluble globulin has been shown to exert a stimulatory effect on the urokinase-mediated activation of bovine plasminogen to plasmin.

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.

A simple, large scale method for preparation of plasminogen-free fibrinogen

Abstract A simple method, involving lysine-Sepharose chromatography was developed for large scale preparation of fibrinogen free from detectable plasminogen. After incubation of the preparation with human urokinase under appropriate conditions for over 24 hours at 37°C, no evidence of degradation of the subunits of fibrinogen was detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Moreover, fibrin clots formed by addition of purified bovine thrombin were not lyzed during incubation for more than 72 hours. Large scale preparation of plasminogen-free fibrinogen requires only a smal column and the treatment takes only a short time, so this method seems much better than those used previously, such as Sephadex G-200 gel filtration or precipitation with ethanol-lysine or ethanol-epsilon aminocaproic acid.

Amino acid sequence of α-bungarotoxin from the venom of bungarus multicinctus

Summary The primary structure of α-bungarotoxin isolated from the venom of Bungarus multicinctus was determined. It composes of 74 amino acid residues including ten half-cystines. Comparing the sequence with those of cobras and sea snake species, striking similarities can be found, especially between α-bungarotoxin and Naja nivea α-toxin, indicating 50 % sequence homology.

Polypeptide chain involved in the cross-linking of stabilized bovine fibrin

Abstract The γ-chain peptide among the three chains of the fibrin molecule participates to the formation of cross-linkages which stabilize the fibrin clot through the action of activated FSF. The cross-linkage in stabilized fibrin may be formed with one or two pairs of γ-chain peptides, because the molecular weight of “X” isolated from S-sulfo-stabilized fibrin seems to be approximately 150,000 to 200,000, judging from the elution volume on a Sepharose 6B column.

Amino acid sequence studies of the fragments produced from horseshoe crab coagulogen during gel formation: Homologies with primate fibrinopeptide B

Summary On incubation of horseshoe crab ( Tachypleus tridentatus ) coagulogen with an endotoxin-activated clotting enzyme, a peptide, named peptide C, is released; the resulting gel protein consists of two chains, name A and B. The complete amino acid sequences of peptide C and A-chain have been established. Their C-terminal octapeptide sequences have significant homology with primate fibrinopeptide B. These facts suggest that the coagulogen and primate fibrinopeptide B are derived from a common ancestor or that the coagulogen is a prototype of primate fibrinogen.

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.

A peptide released from plasma fibrin stabilizing factor in the conversion to the active enzyme by thrombin

Abstract A peptide, which was released accompanying with the activation of bovine plasma fibrin stabilizing factor (FSF) by thrombin, was isolated and characterized. The peptide consisted of Asp4, Thr3, Ser4, Glu4, Pro5, Gly4, Ala4, Val2, Ile1, Leu2, Phe1, and Arg3. The content of proline was highest in all of these amino acids. The carboxyl-terminal residue of the peptide was identified as arginine. However, no N-terminal amino acid reactive with phenyl iso thiocyanate and dansyl chloride could be determined. Edman degradation on the inactive FSF showed glutamic acid or glutamine as one N-terminal residue. After the activation of FSF by thrombin, glycine was identified as a second N-terminal residue, in addition to glutamic acid (glutamine). These results indicate that the transformation of FSF to the active enzyme by thrombin involves proteolysis of an arginyl-glycyl bond located in the N-terminal region of one of the subunits of the proenzyme.

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.