Isolation and functional properties of the heavy and light chains of human plasma kallikrein.
Human plasma kallikrein was prepared by proteolytic activation of prekallikrein with beta-Factor XIIa (Mr = 28,000). Two forms of kallikrein were generated that were each composed of two disulfide-linked polypeptide chains: a heavy chain of apparent Mr = 43,000 and a light chain of apparent Mr = either 36,000 or 33,000. Following reduction and alkylation, the heavy and light chains of kallikrein were isolated by affinity chromatography using insolubilized high molecular weight kininogen. The alkylated light chain of kallikrein did not bind to high molecular weight kininogen-Sepharose while the heavy chain did bind with high affinity and was subsequently eluted. The light chain retained the specific amidolytic activity of native kallikrein. The Km and kcat values for the hydrolysis of H-D-Pro-Phe-Arg-p-nitroanilide by kallikrein or its light chain were identical. Activation of Factor XII in solution was equally well catalyzed by kallikrein and its light chain. However, in kaolin-dependent coagulation, kallikrein was 180 times more effective than the light chain in correcting the clotting defect of prekallikrein-deficient plasma. Furthermore, the light chain was 3.5 times less potent than kallikrein in cleaving high molecular weight kininogen in solution. These observations indicate that the light chain region contains the enzymatic active site and adequately accounts for the enzymatic properties of kallikrein in solution on the protein substrate, Factor XIII, and on oligopeptide substrates. However, the heavy chain region of kallikrein is required for binding to high molecular weight kininogen, for surface-dependent activation of coagulation, and for optimal cleavage of high molecular weight kininogen.
D006801:Humans; D005779:Immunodiffusion; D007610:Kallikreins / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011288:Prekallikrein / Q000097:blood
D046911:Macromolecular Substances; D011288:Prekallikrein; D007610:Kallikreins