Endopeptidase and carboxypetidase activities in human urine which hydrolyze bradykinin

ABSTRACT

We have fractionated the bradykinin inactivating activity of human urine by stepwise elution chromatography on DEAE-cellulose and recovered 95% of the inactivating activity and 29% ofd the protein (absorbance at A280 nm). Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin convertingg activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradyykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides. Cleavage sites demonstrated for Fractioons A through G were Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pho7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G). The relative molecular weight of the bradykininase activity present in each fraction, determined by gel filtration, was 16 kDa (A), 70 kDa (B), 60 kDa (B) (C), 88 kDa (D), 230 kDa (E) and 49 kDa (G). Bradykinin inactiivating activity was inhibited 50--100% by 3 mM EDTA (A,B,D,E adn G), 1 mMM 2-mercaptoethanol (A,B,C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Xn2+ (C), 60 uM BPP5a and 40 uM BPP9a (D), 0.1 uM phosphoramidon (E) and 3 mM sodium p-hydroxyymercuribenzoate (G). The properties of some of these bradykinin inactivating activities correspondend to enzymes previously described in urine and tissues carboxypeptidases (Fractions A and B, angiotensin I converting enzyme (Fraction d), neutral endopeptidase (Fraction E). However, the chymotrypsin-like activity of fractions C and F and the prolylendopeptidase activity of fraction G have not been described before in urine and they being purified in order to obtain a more accurate characterization