Purification of guinea-pig plasma prekallikrein. Activation by prekallikrein activator derived from guinea-pig skin.
Prekallikrein was purified from guinea-pig plasma. The prekallikrein appeared homogeneous as a single-chain protein on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. The apparent molecular weight was 82 000 by SDS-polyacrylamide gel electrophoresis, 99 000 by gel filtration on a Sephadex G-150 column and 84 500 (protein part) by amino acid analysis. The isoelectric point was approx. 9.0. The purification method yielded 3.8 mg (A280 3.800) of prekallikrein from 500 ml of plasma. Kallikrein was generated from the prekallikrein by limited proteolytic action of a prekallikrein activator which was derived from guinea-pig skin. From analysis using SDS-polyacrylamide gel electrophoresis, the kallikrein has two fragments with apparent molecular weights of 52 000 and 40 000 which are linked by disulfide bond(s). The 40 000 molecular weight fragment was shown to incorporate [3H]diisopropylfluorophosphate. The kallikrein hydrolyzed the synthetic substrates containing the Phe-Arg sequence at the COOH-terminal, and it cleaved carbobenzyloxy-Phe-Arg-4-methylcoumaryl-7-amide more readily than Pro-Phe-Arg-methylcoumaryl-7-amide. The Km for the kallikrein with carbobenzyloxy-Phe-Arg-methylcoumaryl amide was 2 times 104 M. Also, the kallikrein showed negligible activities on peptide-methylcoumaryl amide-substrate for alpha-thrombin, Factor Xa or plasmin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004789:Enzyme Activation; D005174:Factor XII / Q000494:pharmacology; D015956:Factor XIIa; D005260:Female; D006168:Guinea Pigs; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000097:blood*; D007705:Kinins / Q000097:blood; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000494:pharmacology; D011288:Prekallikrein / Q000097:blood* / Q000302:isolation & purification; D012867:Skin / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D007705:Kinins; D010446:Peptide Fragments; D005174:Factor XII; D011288:Prekallikrein; D007610:Kallikreins; D015956:Factor XIIa