Hiroshi Mashiko

Cysteine proteinase inhibitors in elapid and hydrophiid snake venoms

The ability of elapid and hydrophiid snake venoms to inhibit cathepsin L was tested. All nine species of elapid and three species of hydrophiid snake venoms tested showed inhibition against cathepsin L. All of these venoms tested also showed inhibition against papain as well as against cathepsin L. Among these venoms, two elapid (Laticauda semifasciata venom, and Ophiophagus hannah venom) and one hydrophiid snake venom (Notechis scutatus scutatus venom) showed strong inhibition against both cathepsin L and papain. These venoms contained 12.0-13.0 kDa low molecular-weight cysteine proteinase inhibitors.

Studies on factor X11 in porcine plasma: purification and its conversion to activated form by porcine plasma kallikrein

When porcine plasma was subjected to four steps of ion-exchange column chromatographies, factor XII (F. XII) was separated into two fractions, F. XII-1 and F.XII-2, and about 8.5 mg of F. XII-1 and 2.3 mg of F. XII-2 were obtained from 675 ml of the plasma. The purified proteins were found to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular masses of F. XII-1 and XII-2 were estimated to be about 84 kDa by SDS-PAGE, and consisted of a single polypeptide chain. However, F. XII-1 converted to active form after removing Polybrene, but F. XII-2 was not activated. Activation of F. XII-2 took place on incubation with porcine plasma kallikrein, and the activation rate was increased only in the presence of negatively charged surfaces, such as sulfatide. When the preparation was incubated with sulfatide, spontaneous activation was not observed on the condition that we used. Activation of porcine F. XII-2 by porcine plasma kallikrein was found to involve the cleavage of the peptide bond on the disulfide-bridged polypeptide chain, and further fragmentation of activated form was observed during prolonged incubation time.

Haemorrhagic Factors from Snake Venoms II. Structures of Haemorrhagic Factors and Types and Mechanisms of Haemorrhage

AbstractIt was revealed that almost all snake venom haemorrhagic factors (HFs) are metalloproteinases. Analysis of primary structures of HFs revealed that they share multi-domain structures. And they are divided into four major classes. These HFs damaged the micro blood vessel walls and cause local haemorrhage. Some HFs cause systemic, organ specific and species specific haemorrhage. This review describes the structures of HFs and autoproteolysis of HFs. Types and mechanisms of haemorrhage caused by HFs are also described.

Canine Plasma Kininogen: Evidence for the Presence of Two Kininogens and Purification of High Molecular Weight Kininogen and Characterization as Multi-Functional Molecule*

The ratio of kininogen that is substrate of plasma kallikrein to kininogen, which is not substrate of plasma kallikrein in canine plasma, was about 1:3.6 by differential assay of kininogens. When the plasma was gel-filtered through a column of Sephacryl S-300 superfine, two fractions, which released kinin by trypsin, were obtained. These results indicate that two kininogens with different molecular weights are present in the plasma and they show different susceptibility to plasma kallikrein. One kininogen was purified by ion-exchange and zinc-chelating affinity chromatographies. Purified kininogen showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition and its molecular weight was 125 kDa. Released kinin from the kininogen by trypsin was bradykinin. The kininogen inhibited papain and ficin but did not inhibit bromelain at the concentration used. The kininogen bound to carboxymethylated-papain and this binding was dissociated by 3M NaSCN. Canine plasma shortened the abnormal clotting time of human high molecular weight kininogen-deficint plasma. The kininogen also shortened the abnormal clotting time of the plasma. From these results, the purified kininogen was high molecular weight kininogen and it was multi-functional protein.

Studies on porcine high molecular weight kininogen. An improved method for the purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein

By introduction of stepwise DEAE Sephadex A-50 and copper-Chelating Sepharose 6B column chromatographies, about 18.5 mg of high molecular weight kininogen (HK) composed of a single polypeptide chain was obtained from 500 ml of porcine plasma. Molecular weights of reduced or non-reduced preparation were estimated to be 110 kDa and 116 kDa, respectively, by SDS-PAGE. Using the preparation, cleavage of HK by porcine plasma kallikrein (KK) was investigated. A single polypeptide HK was cleaved into two chains cross-linked by disulfide bond(s), accompanying the release of kinin. Further degradation was not observed. Molecular weights of heavy-chain (H-chain) and light-chain (L-chain) were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino- (N-) terminal sequences of intact HK, reduced and carboxymethylated- (RCM-) H-chain, RCM-L-chain and the peptide around the kinin moiety obtained by BrCN digestion were determined. Their sequences were highly homologous with those of bovine or human HK. These results indicate that plasma KK first cleaved the Arg-Ser bond of HK, and formed nicked HK. The second cleavage yielded bradykinin (BK) and kinin-free protein, which was apparently of equal size to the nicked HK. The structure of HK was from the N-terminus to the carboxy- (C-) terminus, H-chain-BK-L-chain.

Haemorrhagic Factors from Snake Venoms. I. Properties of Haemorrhagic Factors and Antihaemorrhagic Factors

AbstractHaemorrhagic factors were purified from many snake venoms belong to Crotalid and Viperid snakes, and they were characterized. These results indicate that they are metalloproteinases, and divided into four groups by their molecular weights and domain compositions, such as small(15∼30 kDa), medium (30∼50 kDa), large (50∼80 kDa) and extra large (>80 kDa). And their enzymatic properties were differed from each other. Antihaemorrhagic factors were purified from the serum of venomous, non-venomous and warm-blooded animals, and characterized. In this review, we describe the properties of these haemorrhagic factors and antihaemorrhagic factors.

Bullfrog plasma cysteine proteinase inhibitor (CPI)

Papain is inhibited by bullfrog plasma. CPI was isolated from bullfrog plasma by two step column chromatography; Cm-papain agarose column chromatography followed by FPLC Mono Q column chromatography with addition of benzamidine. Isolated CPI gave a single band on SDS-PAGE under reducing condition, and the molecular weight of reduced CPI was estimated to be over 200 kDa. When the isolated CPI was stored at 4 degrees C in the absence of benzamidine, the CPI was cleaved and yielded a protein consisting of heavy chain (60 kDa) and light chain (54 kDa). Both chains were linked with disulfide bond(s). The cleaved form of CPI was also isolated from the plasma in the absence of benzamidine. Intact and cleaved form of CPI inhibited papain and ficin, but not trypsin.

Cleavage of porcine high molecular weight kininogen by the action of porcine pancreatic kallikrein.

Pancreatic kallikrein (KK), converts the single chain high molecular weight kininogen (HK) into a two-chain molecule accompanied by kinin release. Further degradation was not observed. Molecular weights of the heavy (H)-chain and light (L)-chain were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino (N)-terminal sequences of intact HK, reduced and carboxymethylated (RCM)-H-chain, and RCM-L-chain were determined. These results indicate that porcine pancreatic KK initially cleaved the Arg-Ser bond of HK, to form a two chain molecule. Then pancreatic KK cleaved the Met-Lys bond yielding kallidin and a kinin-free protein, which was apparently equal in size to the nicked kininogen.