Kinin-converting aminopeptidase from human serum
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Data
1973-01-01
Autores
Guimarães, Jorge A. [UNIFESP]
Borges, Durval R. [UNIFESP]
Prado, Eline S. [UNIFESP]
Prado, José L. [UNIFESP]
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Resumo
A kinin-converting aminopeptidase from human serum (HuPA) which converts kallidin (lysylbradykinin, LBK) to bradykinin (BK) was purified about 900-fold by a three-step procedure involving ammonium sulfate fractionation, continuous loading Sephadex gel electrophoresis, and gel filtration on Sephadex G-200; the final yield was about 7 per cent. A bioassay was developed in which BK formed in the presence of excess LBK or methionyllysylbradykinin (MLBK), pH 7.8, 37°, was separated on carboxymethylcellulose columns and assayed on the isolated guinea pig ileum. L-Aminoacyl-β-naphthylamidase activities of HuPA measured on L-lysine, L-arginine- and L-leucylnaphthylamides (Lys-NA, Arg-NA and Leu-NA) were parallel to the kinin-converting activity in all purification steps. The activity of the purest preparation on LBK and MLBK, measured at the approximate enzyme:substrate molar ratio 1:1500, was respectively 267 and 174 nmoles BK/min/mg of protein. Considering the activity on Met-NA as 100, the activities on other naphthylamides were: Leu-NA, 65; Arg-NA, 46; Lys-NA, 29; Asp-NA, 0; Glu-NA, 0. A few tri- and dipeptides, angiotensin II and its amide, polylysine and polyarginine were poorer substrates. The following substances were inhibitors: 1,10-phenanthroline, puromycin, EDTA and 2,3-dimercapto-l-propanol. 1,10-Phenanthroline or EDTA inhibition could be partially reversed by Co2+, Mn2+ and Zn2+. The molecular weight was estimated as 95,000 on Sephadex gel filtration. On agarose gel microelectrophoresis, the enzyme migrated as α1-globulin before the electrophoresis step and had a migration intermediate between α2- and β-globulin following this step. In this microelectrophoresis, only one band of arylamidase activity was detected on Lys-, Arg- and Leu-NA. The purest preparation was still contaminated with albumin but was free of kininase activity.
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Biochemical Pharmacology. Oxford: Pergamon-Elsevier B.V., v. 22, n. 24, p. 3157-3172, 1973.