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.
Mashiko
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Division of Chemistry of Hygiene, Meiji College of Pharmacy, Tokyo, Japan.
Miyamoto
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Takahashi
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007610:Kallikreins / Q000378:metabolism*; D019679:Kininogen, High-Molecular-Weight / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013552:Swine / Q000097:blood* / Q000235:genetics
D000596:Amino Acids; D019679:Kininogen, High-Molecular-Weight; D007610:Kallikreins