Human plasma kallikrein. Immunoreactivity and activity on natural and synthetic substrates.
Human plasma kallikrein (HuPK) is a serine protease found in mammalian plasma. Following limited proteolysis, the enzyme is activated and forms two chains. The light chain occurs with molecular weight 36,000 or 33,000 and contains the active site. The heavy chain occurs with molecular weight of 45,000 and contains the binding site for high-molecular-weight kininogen. Both chains were prepared from active HuPK, following mild reduction with dithiothreitol and carboxymethylation with iodoacetamide. The light chain was isolated in SBTI-Sepharose and its kinetic properties were determined with synthetic derivatives of arginine-p-nitroanilides, to investigate any possible alteration of the active site. These studies showed that substrate modifications affected the hydrolytic activity more than the binding capacity. The ability to cleave high-molecular-weight kininogen, as observed for intact kallikrein, was reduced in beta-kallikrein and absent in the light chain, even when equimolar amounts of both light and heavy chains were tested together. Anti-kallikrein antiserum formed immunoprecipitates not only with kallikrein itself, but also with the separated chains. The immunoreactivity of the light-chain was not identical with that of kallikrein. Immunoselected specific antibodies for both chains, depending on the selectivity, reacted with the heavy chain and kallikrein, or with the light chain and kallikrein. These antibodies were shown to be effective in binding radio-iodinated HuPK in the radioimmunoassay developed for intact kallikrein.
Motta
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G
Department of Biochemistry, Escola Paulista de Medicina, São Paulo, Brazil.
Sampaio
C A
CA
Sampaio
M U
MU
D000595:Amino Acid Sequence; D000906:Antibodies / Q000032:analysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D007610:Kallikreins / Q000097:blood* / Q000276:immunology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011863:Radioimmunoassay
D000906:Antibodies; D007610:Kallikreins