Characterization of kininogenase activity of an acidic proteinase isolated from human kidney

Characterization of kininogenase activity of an acidic proteinase isolated from human kidney

Author Gomes, RAS Google Scholar
Juliano, L. Autor UNIFESP Google Scholar
Chagas, JR Autor UNIFESP Google Scholar
Hial, V Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract An acidic proteinase was purified from human kidney cortex. the enzyme showed a molecular mass of 31 kDa by SDS-PAGE, 36 kDa by gel filtration, and isoelectric points of 5.2 and 6.1. the optimum pH for hydrolysis of bovine hemoglobin was about 3.5. Reverse-phase KPLC analysis of the incubation mixture of the enzyme with human plasma showed the presence of an active peptide on rat uterus muscle with the same retention time as the methionyl-lysyl-bradykinin (MLBK) standard. the specific activities were 2.91 mu g MLBK equivalent.mg(-1).min(-1) at pH 3.5 and 2.15 mu g MLBK equivalent.mg(-1).min(-1) at pH 6.0. All the enzymatic activities of this human kidney proteinase were inhibited by pepstatin A. Intramolecularly quenched fluorogenic substrates with amino acid sequences of human kininogen were used to determine the cleavage points. On the N-terminal sequences (Abz-Leu-Met-Lys-Arg-Pro-Eddnp and Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp) the cleavage occurred at the Leu-Met linkage, and on the C-terminal sequences (Abz-Phe-Arg-Ser-Ser-Arg-Eddnp and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp) the cleavage occurred at the Arg-Ser linkage. Abz-Arg-Pro-Pro-GIy-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp was hydrolyzed by the renal acidic proteinase and yielded the peptide Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (Abz-bradykinin). Kinetic parameters were determined using Abz-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Eddnp (K-m=0.69+/-0.08 mu M; K-cat=0.052+/-0.0095 s(-1); K-cat/K-m= 0.075+/-0.005 mu M-1.s(-1)) and Abz-Phe-Arg-Ser-Ser-Arg-Gln-Eddnp (K-m=1.56+/-0.16 mu M; K-cat=0.0048+/-0.0001 s(-1); K-cat/K-m=0.003+/-0.0003 mu M-1.s(-1)). Human liver cathepsin D had no activity on C-terminal sequences and human pepsin hydrolyzed them at the Ser-Ser bond. the results suggest that the renal acid proteinase is distinct from human pepsin and human liver cathepsin D and releases MLBK from human kininogen.
Keywords kininogenase
acidic proteinase
kinin
Language English
Date 1997-06-01
Published in Canadian Journal of Physiology and Pharmacology. Ottawa: Natl Research Council Canada, v. 75, n. 6, p. 757-761, 1997.
ISSN 0008-4212 (Sherpa/Romeo, impact factor)
Publisher Natl Research Council Canada
Extent 757-761
Origin http://dx.doi.org/10.1139/cjpp-75-6-757
Access rights Open access Open Access
Type Article
Web of Science ID WOS:A1997XR50500036
URI http://repositorio.unifesp.br/handle/11600/25730

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