Akira Ohsaka

Purification and some properties of two hemorrhagic principles (HR2a and HR2b) in the venom of Trimeresurus flavoviridis; complete separation of the principles from proteolytic activity.

Abstract 1. 1. One of the hemorrhagic fractions (HR2), containing the bulk of proteolytic activity, separated from the venom of Trimeresurus flavoviridis was further purified. The hemorrhagic activity was determined quantitatively by the well-defined skin reaction and the parallel-line assay. Chromatography on Bio-Rex 70 resolved the hemorrhagic activity into two parts, HR2a and HR2b; both were completely separated from the proteolytic activity. The results may suggest that the hemorrhage in snake bite is not due to the proteolytic enzyme in the venom. 2. 2. Twelve-fold purification was achieved with a yield of 25% with respect to HR2a and 13-fold with a yield of 12% with respect to HR2b. In ultracentrifugation and cellulose acetate electrophoresis, HR2a behaved as a homogeneous protein but HR2b did not, probably being contaminated with HR2a. HR2a had an s20,w of 2.4 S and HR2b approx. 2.0 S. These principles shared a common susceptibility to heat, pH and such reagents as EDTA or cysteine.

Purification and characterization of an antihemorrhagic factor in the serum of Trimeresurus flavoviridis, a crotalid

Abstract 1. 1. An antihemorrhagic factor in the serum of Trimeresurus flavoviridis, a crotalid, was purified by (NH4)2SO4 fractionation, ethanol fractionation, gel filtration on Sephadex G-200, chromatography on DEAE-cellulose and isoelectric focusing in carrier ampholytes. A 21-fold increase in specific activity was attained with a yield of 15%. The purified preparation was homogenous as judged by ultracentrifugation, gel filtration on Sephadex G-200, disc electrophoresis, immunoelectrophoresis and isoelectric focusing. 2. 2. The purified serum factor inhibited the two immunologically distinct hemorhagic principles, HR1 and HR2 (mixture of HR2a and 2b), in the venom of this snake and antihemorrhagic activities against HR1 and HR2 increased simultaneously during purification. The purified factor inhibited not only the hemorrhagic activities but also the lethal toxicity in the venom. 3. 3. The molecular weight of the purified factor was approx. 70 000 (s°20,w = 4.05); the isoelectric point was around 4. The purified factor migrated to a position in the area of albumin and α1-globulin in immunoelectrophoresis and did not form any precipitin line with the crude venom or with the purified hemorrhagic principles in immunodiffusion tests. The relation of the serum factor to immunoglobulins was discussed. 4. 4. The crude serum from T. flavoviridis inhibited hemorrhagic activities of all snake venoms tested, including Crotalidae, Viperidae and Elapidae venoms. A possible mechanism of the inhibition by the serum factor of the hemorrhagic activity of snake venoms was also discussed.

Purification and characterization of a proteinase in the venom of Trimeresurus flavoviridis: Complete separation of the enzyme from hemorrhagic activity

Abstract 1. 1. A proteinase was purified from the venom of Trimeresurus flavoviridis, by gel filtration on Sephadex G-100 and column chromatography on Amberlite CG-50 (Type 2), with a 27% over-all yield and a 9-fold increase in specific activity. This enzyme, named H2-proteinase, is the major proteinase in the venom. 2. 2. The purified enzyme was completely free from hemorrhagic activity. It was proven that large amounts of this enzyme had contaminated all preparations of Hemorrhagic Principle 2 so far obtained. 3. 3. The purified enzyme was proved to be homogeneous by ultracentrifugation, sucrose-density-gradient centrifugation, electrophoresis on cellulose acetate membrane and from the absence of any of the other enzyme activities detected in the original venom. 4. 4. H2-proteinase had an s 20,w of 2.43 S and a molecular weight of 24 000, as shown by the sedimentation velocity and the approach-to-equilibrium methods. The enzyme was most active at pH 9.5 with casein as substrate and the enzyme activity was abolished completely by addition of Cd2+, Ni2+, EDTA or cysteine. 5. 5. H2-proteinase split the oxidized B chain of insulin at the three peptide bonds, Asn3Gln4, His10Leu11 and Ala14Leu15. Of the synthetic leucine-containing peptides of varying sizes tested, the longest one, ZGlyProLeuGlyPro·OH, alone was split by the enzyme at the peptide bond ProLeu. A certain dependence of the enzymatic hydrolysis on the chain length of the substrate was suggested.

Purification of Clostridium perfringens phospholipase C (α-toxin) by affinity chromatography on agarose-linked egg-yolk lipoprotein

Abstract 1. 1. Clostridium perfringens phospholipase C (α-toxin) (EC 3.1.4.3) was extensively purified by affinity chromatography on agarose-linked egg-yolk lipoprotein, followed by gel filtration on Sephadex G-100. The purification achieved was 200-fold from crude enzyme (2500-fold from the culture filtrate) with a yield of approx. 60% with respect to either enzymatic, lethal or hemolytic activity. 2. 2. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis, ultracentrifugation and immunodiffusion. The molecular weight estimated was 43 000. The purified preparation had the highest specific activity among the preparations ever reported. 3. 3. By isoelectric focusing of the purified enzyme, three major molecular forms, designated as α 0 -, α 1 - and α 2 -toxins, and several minor ones were isolated. Isoelectric points for α 0 -, α 1 - and α 2 -toxins were 5.2, 5.3 and 5.5, respectively. These multiple forms were not distinguished from each other by immunodiffusion and electrophoresis in polyacrylamide gel with or without sodium dodecylsulfate. They shared a common susceptibility to inactivation by heat and extreme pH. 4. 4. All the multiple forms isolated had phospholipase C activity towards both lecithin and sphingomyelin, as well as lethal and hemolytic activities, indicating that these three activities reside in a single entity.

Purification and Some Properties of Hemorrhagic Principle I in the Venom of Trimeresurus Flavoviridis

Summary 1. One of the hemorrhagic principles, hemorrhagic principle I, in the venom of Trimeresurus flavoviridis was highly purified by gel filtration on Sephadex G-100, by chromatography on DEAE-Sephadex A-50 and on guanidoethyl cellulose and by recycling chromatography on Sephadex G-200. The hemorrhagic activity was determined quantitatively by the well-defined skin reaction and the parallel-line-assay. A 43-fold increase in specific activity was attained with a yield of 20% with respect to hemorrhagic activity. The minimum hemorrhagic dose of the purified preparation was 0.0058 βg. Thus, the preparation possessed the highest hemorrhagic activity ever purified among the hemorrhagic principles of bacterial and animal origins. It also contained potent lethal toxicity (L.D.50 = 4.63 ftg per mouse). 2. The preparation was homogeneous as judged by electrophoresis on cellulose acetate membrane, isoelectric focusing and immunodiffusion but not homogeneous by ultracentrifugation. The hemorrhagic principle had a molecular weight of approx. 100 000 (s20,w = 5.8 S) and an isoelectric point of 4.3. The hemorrhagic activity was inhibited by such reagents as EDTA, cysteine or formaldehyde but not by DFP or soybean trypsin inhibitor. The preparation contained 7% of the lethal toxicity and 0.6% of the proteolytic activity present in the original venom. Possible relations of the hemorrhagic activity to the lethal toxicity and to the proteolytic activity are discussed.

A simple and rapid method for separating two hemorrhagic principles in the venom of Trimeresurus flavoridis

Abstract A new method was presented for separating on a large scale two hemorrhagic principles in the venom of Trimeresurus flavoviridis. The method was based on molecular sieving on Sephadex G-100 of crude venom. One of the two principles prepared by this method was proved to be identical with Hemorrhagic Principle 1 (HR1) and the other with HR2 in respect to electrophoretical mobility and immunological specificity. HR1 and HR2 are the principles obtained by fractionating crude venom by zone electrophoresis as reported previously. The present method is simple and rapid and apparently facilitates preparation of individual hemorrhagic principles in amounts sufficiently large for use in separate titrations of anti-HR1 and anti-HR2 in an antivenine and further purification of the principles.

Vascular permeability increase by α-toxin (phospholipase C) of Clostridium perfringens

T. Sugahara, T. Takahashi, S. Yamaya and A. Ohsaka. Vascular permeability increase by α-toxin (phospholipase C) of Clostridium perfringens. Toxicon15, 81–87, 1977.—Highly purified α-toxin increased the vascular permeability in guinea-pig skin. This vascular permeability-increasing activity reached a minimum value at around 70°C but heating at higher temperatures inactivated it to a lesser extent. The same anomaly in heat inactivation was observed with the phospholipase C activity possessed by the toxin. By subjecting the purified toxin to isoelectric focusing, four molecular forms were isolated, all of which were associated with both vascular permeability-increasing and enzymatic activities. These results clearly demonstrated the identity of the factor responsible for the vascular permeability-increasing activity with α-toxin.

Aggregation of platelets in the mesenteric microcirculation of the rat induced by α- toxin (phospholipase C) of Clostridium perfringens

Abstract We have investigated the effects of purified α-toxin (phospholipase C) of Clostridium perfringens ( Yamakawa and Ohsaka , 1977) on the behavior of cellular components in the microcirculation, using cinematography on a microscopic level and electron microscopy. We demonstrated that after topical application of α-toxin to the mesentery of the rat, rolling of leucocytes along the vessel wall and sticking of leucocytes to the vessel wall occurred in venules but not in arterioles. Some of the leucocytes remained attached to the vessel wall. Thrombi were formed frequently in venules and capillaries, and at a later stage, in arterioles. With time, thrombi increased in number and size, leading eventually to stasis of the blood stream. Thrombi were also observed frequently in the mesenteric microcirculation when toxin was injected into the jugular vein of the rat. The experiments with adenosine pretreatment followed by topical application of α-toxin to the mesentery suggested that the formation of thrombi induced by this toxin does not involve the mediation of ADP. Electron-microscopic examination confirmed the formation of thrombi consisting solely of platelets. It was concluded that thrombosis must be involved as an early step in the pathogenesis of necrosis caused by α-toxin. The death of the animals injected intravenously with α-toxin may be due, at least in part, to thrombosis. It is possible that thrombosis induced by α-toxin may be one of the factors involved in the causation of toxemia often manifested in the late stage of gas gangrene.

1H- and 13C-NMR spectroscopic study of the metabolites in young adult Angiostrongylus cantonensis maintained in vitro

1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy were used to study metabolites excreted by young adult Angiostrongylus cantonensis maintained aerobically in the presence of D-[13C6]glucose. End-products of glucose metabolism identified and quantitated by means of 1H-NMR were lactate, acetate and alanine, in the molar ratio of 1:0.13:0.05 for males and 1:0.07:0.04 for females. 13C-NMR analyses proved that all the three products originated from the glucose present in the medium.

Identification and quantitation by 1H-NMR of metabolites in animal organs and tissues. An application of NMR spectroscopy in forensic science

1H-nuclear magnetic resonance (NMR) was applied to the study of metabolites in rat organs and tissues. From 1H-NMR spectra of D2O extracts of various organs and tissues, lactate, pyruvate, and some other substances were simultaneously identified and quantitated. On the basis of the results, the use of 1H-NMR for estimating post-mortem time was suggested.