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