Mashiko H, Miyamoto K, Takahashi H
Division of Chemistry of Hygiene, Meiji College of Pharmacy, Tokyo, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 1998 Aug;120(4):647-56. doi: 10.1016/s0305-0491(98)10057-3.
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.
通过逐步引入二乙氨基乙基葡聚糖A-50和铜离子螯合琼脂糖6B柱色谱法，从500毫升猪血浆中获得了约18.5毫克由单一多肽链组成的高分子量激肽原（HK）。通过SDS-PAGE估计，还原或非还原制剂的分子量分别为110 kDa和116 kDa。使用该制剂，研究了猪血浆激肽释放酶（KK）对HK的裂解作用。单一多肽HK被裂解为通过二硫键交联的两条链，同时释放出激肽。未观察到进一步的降解。通过SDS-PAGE估计，重链（H链）和轻链（L链）的分子量分别为61 kDa和56 kDa。测定了完整HK、还原和羧甲基化的（RCM-）H链、RCM-L链以及通过溴化氰消化获得的激肽部分周围肽段的氨基（N-）末端序列。它们的序列与牛或人HK的序列高度同源。这些结果表明，血浆KK首先裂解HK的精氨酸-丝氨酸键，形成带切口的HK。第二次裂解产生缓激肽（BK）和无激肽蛋白，其大小显然与带切口的HK相等。HK的结构从N末端到羧基（C-）末端为H链-BK-L链。
