title: Specificity comparison of a serine endopeptidase (SH1) and a serine thiol endopeptidase (STH2) purified from human urine.
abstract: In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].
authors.affiliation: Departamento de Medicina, Disciplina de Nefrologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 740, CEP 04023-900, São Paulo, SP, Brazil.
authors.forename: B M R
authors.initials: BM
authors.lastname: Quinto
authors.forename: M A
authors.initials: MA
authors.lastname: Juliano
authors.forename: I
authors.initials: I
authors.lastname: Hirata
authors.forename: A K
authors.initials: AK
authors.lastname: Carmona
authors.forename: L
authors.initials: L
authors.lastname: Juliano
authors.forename: D E
authors.initials: DE
authors.lastname: Casarini
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism / Q000652:urine*; D013379:Substrate Specificity
chemical_list: D012697:Serine Endopeptidases