Characterization of a kinin inactivating serine endopeptidase H2 (kininase) from human urine using fluorogenic substrates.
We have previously described a kinin-inactivating endopeptidase (H2), which was purified 19-fold from human urine by DEAE-cellulose chromatography and gel filtration. The enzyme was inhibited 100% by PMSF, TPCK and pOHMB. In the present communication, we further characterized this enzyme using the fluorogenic substrates Abz-RPPGFSPFRQ-EDDnp (Abz-BKQ-EDDnp) and Abz-FRQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl] ethylenediamine). Also a rapid, sensitive and specific assay for the H2 was developed. The enzyme hydrolyzed bradykinin (BK = RPPGFSPFR) at the F-S peptide bond, differing from the cleavage site F-R, in the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FRQ-EDDnp. Other enzymes present in urine as the serine endopeptidase H1, prolyl endopeptidase and neutral endopeptidase-like were not able to hydrolyze the related substrate Abz-FRQ-EDDnp. The determined Km for Abz-BKQ-EDDnp and Abz-FRQ-EDDnp were 0.79 microM and 3.02 microM, respectively. Using the fluorogenic substrates, we observed that PMSF and p-hydroxymercuribenzoate irreversibly inhibited the enzyme H2. E-64 was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. The inhibition observed in the presence of pOHMB was partially reversed by 2 mM cysteine. These results suggest that the H2 enzyme belongs to the subfamily of SH-containing serine proteases. Based on the molecular weight of isolated H2 (60 kDa), we believe that this enzyme originated from the kidney and may cleave the kinins filtered through the glomerulus and also that produced in the kidney.
Quinto
B M
BM
Depto. de Medicina, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil.
Juliano
L
L
Juliano
M
M
Carmona
A K
AK
Stella
R C
RC
Casarini
D E
DE
D001920:Bradykinin; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism / Q000652:urine*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007705:Kinins / Q000378:metabolism*; D010446:Peptide Fragments; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D007705:Kinins; D010446:Peptide Fragments; D010450:Endopeptidases; D012697:Serine Endopeptidases; D001920:Bradykinin