Nobuhiro Mori

Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (Western Diamondback Rattlesnake)

Hemorrhagic toxin f (HT-f) was isolated from Crotalus atrox (Western Diamondback Rattlesnake) venom by a five-step purification procedure. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing, and sodium dodecyl sulfate (SDS)-electrophoresis. HT-f has a molecular weight of 64,000 and contains 572 amino acid residues. It contains 1 mol of zinc per mol of protein. Zinc is essential for both hemorrhagic and proteolytic activities. HT-f possesses proteolytic activity hydrolyzing the Val-Asn, Gln-His, Leu-Cys, His-Leu, Ala-Leu, and Tyr-Leu bonds of oxidized insulin B chain. HT-f did not coagulate fibrinogen to fibrin, yet it did hydrolyze the gamma chain of fibrinogen without affecting either the A alpha or B beta chains. This is the first time that a hemorrhagic toxin was shown to have fibrinogenase activity. HT-f was shown to differ immunologically from other hemorrhagic toxins such as HT-a and HT-c. HT-f also possesses lethal toxicity. When zinc was removed the apo-HT-f lost its lethal toxicity. HT-f produced not only local hemorrhage in the skin and muscle, but also produced systemic hemorrhage in internal organs such as the intestine, kidney, lung, heart, and liver.

Isolation and characterization of hemorrhagic factors a and b from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.

Purification of a proteinase (Ac5-proteinase) and characterization of hemorrhagic toxins from the venom of the hundred-pace snake (Agkistrodon acutus)

Ac5-Proteinase (15.2 mg) was isolated from Agkistrodon acutus venom (1 g) by column chromatography on Sephadex G-75, CM-Sephadex C-50 and CM-Cellulose. Ac5-Proteinase was homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3 and also by SDS-disc polyacrylamide gel electrophoresis. Ac1-, Ac2-, Ac3- and Ac5-proteinases possessed lethal and hemorrhagic activities, but Ac4-proteinase had no lethal activity. These activities were inhibited completely by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine. The molecular weights of Ac1-, Ac2-, Ac3-, Ac4- and Ac5-proteinases were approximately 24,500, 25,000, 57,000, 33,000 and 24,000, respectively. Ac1-, Ac2-, Ac4- and Ac5-proteinases did not contain any carbohydrates, but Ac3-proteinase contained 0.1% carbohydrate by weight.

Characterization of mucrotoxin a from the venom of Trimeresurus mucrosquamatus (the chinese habu snake)

Mucrotoxin A from the venom of Trimeresurus mucrosquamatus was isolated in homogeneous form by a previously published method. Mucrotoxin A did not hydrolyze casein, however, when dimethylcasein was used as a substrate, the toxin cleaved the substrate. This toxin also hydrolyzed the oxidized B chain of insulin and fibrinogen. The sites of cleavage in the oxidized B chain of insulin were identified as Ser(9)-His(10), His(10)-Leu(11), Ala(14)-Leu(15), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The toxin digested the A alpha chain of fibrinogen first, followed by hydrolysis of the B beta chain. The fact that no fibrin clot formed indicates that the sites of cleavage in the A alpha and B beta chains of fibrinogen by the toxin must be different from those cleaved by thrombin. Mucrotoxin A produced systemic hemorrhage in internal organs such as the heart and stomach.

Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase.

A new type of fibrinogenase was isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Unlike thrombin, the newly isolated fibrinogenase did not cause formation of a fibrin clot. Various properties of the fibrinogenase we isolated were compared with crotalase isolated from the venom of C. adamanteus. It was found that fibrinogenase has considerable similarity to crotalase isolated by Markland and Damus in 1971. Crotalase is a thrombin-like enzyme and produces a fibrin clot from fibrinogen. The A alpha chain of fibrinogen was first split and the B beta chain was cleaved later. The fact that no fibrin clot forms indicates that the cleavage sites in A alpha and B beta chains of fibrinogen must be different from thrombin sites. The fibrinogenase also released bradykinin by interacting with plasma proteins. It hydrolyzed TAME (p-toluenesulfonyl-L-arginine methyl ester), BAEE (N-benzoyl-L-arginine ethyl ester). TLME (N-tosyllysine methyl ester) but not BAA (N-benzoylarginine amide), TAA (N-tosylarginineamide) or ATEE (N-acetyltyrosine ethyl ester). The enzyme is an acidic protein with pI of 4.6 and a mol. wt of 31,000. It consists of 272 total amino acid residues, 21% of which are acidic amino acids. Fibrinogenase is a specific form of protease. A newly liberated amino group after hydrolysis of dimethyl-casein can be detected by the reagent trinitrobenzenesulfonic acid (TNBS). Fibrinogenase differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzymes action.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of three proteinases from the venom of the Chinese habu snake (Trimeresurus mucrosquamatus)

Three immunochemically distinct proteinases (P-1, 2 and 3) devoid of hemorrhagic activity were isolated from the lyophilized venom of Trimeresurus mucrosquamatus using column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel, CM-Cellulose and Bio-Rex 70. By these procedures, about 7.6, 7.3 and 8.2 mg of purified P-1, 2 and 3 may be obtained from 1 g of crude venom, respectively. The purified proteinases 1-3 were homogeneous by disc electrophoresis on polyacrylamide gel at pH 4.3, isoelectric focusing and by the presence of one precipitin line on immunodiffusion. The isoelectric point of P-1 was 8.1; P-2, 9.2; P-3, 9.8. The molecular weights of proteinases 1-3 were determined to be 23,000, 23,500 and 23,000, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, respectively. The purified proteinases 1-3 possessed caseinolytic and fibrinogenolytic activities. These activities were inhibited when the proteinases were incubated with the metal chelators ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or cysteine, but not with egg white trypsin inhibitor (EWTI) or soybean trypsin inhibitor (SBTI). P-1 cleaved the B beta-chain of fibrinogen first and then the A alpha-chain, whereas P-2 and 3 cleaved the A alpha-chain first and then the B beta-chain. However, these three proteinases did not hydrolyze the gamma-chain.

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. Specific esterolytic activities of the kallikrein-like enzyme on N-tosyl-L-arginine methylester (TAME) and N-benzoyl-L-arginine ethylester (BAEE) are 109.5 and 23.6 mumol/min/mg, respectively. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzymes action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. The enzyme is not lethal at 15 micrograms/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.

Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. A kallikrein-like enzyme from the venom of Crotalus ruber ruber (red rattlesnake) had been isolated and characterized by Mori and Sugihara. The enzyme was active upon the kallikrein substrates, Pro-Phe-Arg-MCA and z-Phe-Arg-MCA, and slightly hydrolyzed Boc-Val-Leu-Lys-MCA, and Boc-Phe-Ser-Arg-MCA. 2. Unlike thrombin, the newly isolated kallikrein-like enzyme did not cause formation of a fibrin clot when fibrinogen was mixed with the enzyme. 3. The B beta chain of fibrinogen was first split and A alpha chain was cleaved later. Pancreatic kallikrein hydrolyzed only the A alpha chain without affecting the B beta chain. 4. The kallikrein-like enzyme produced kallidin (Lys-bradykinin) by splitting the Met-Lys bond instead of producing bradykinin. 5. The kallikrein analog JSI-450 (Ac-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser-NH2) was also cleaved at the site of the Arg-Ser bond. 6. Its NH2-terminal amino acid sequence (Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-Arg-Pro-Phe-Leu-Val-Ala-Leu-Tyr- Asp-Ser-) is homologous to the rat pancreatic kallikrein and other snake venom proteases.

Comparative study of two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber (red rattlesnake).

1. Two arginine ester hydrolases, E-I and E-II from the venom of Crotalus ruber ruber were isolated and characterized. 2. E-I and E-II have molecular weights of 32,000 and 33,000, and isoelectric points of 5.2 and 4.6, respectively. 3. E-I and E-II are active upon the glandular kallikrein substrate, but neither enzyme was shown to have plasma kallikrein substrate hydrolytic activity. 4. E-I has minimal fibrinogen-clotting activity. It was found to induce clotting by catalyzing the hydrolysis of only the A fibrinopeptide from the A alpha-chain of fibrinogen.