ENDOPEPTIDASE AND CARBOXYPEPTIDASE ACTIVITIES IN HUMAN URINE WHICH HYDROLYZE BRADYKININ

1. 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% of the protein (absorbance at A280 nm).2. Seven of nine fractions which presented activity were also tested for angiotensin I and II inactivating activity, angiotensin converting activity and for the hydrolysis of hippuryl-His-Leu and hippuryl-Arg. Sites of hydrolysis in bradykinin were determined by HPLC of the hydrolysates and fragments were compared with authentic peptides.3. Cleavage sites demonstrated for Fractions A through G were: Phe8-Arg9 (A and B), Phe5-Ser6 (C and F), Pro7-Phe8 (D), Gly4-Phe5 and Pro7-Phe8 (E) and Pro3-Gly4 (G).4. 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 (C), 88 kDa (D), 230 kDa (E), 45 kDa (F) and 49 kDa (G).5. Bradykinin inactivating activity was inhibited 50-100% by 3 mMEDTA (A,B,D,E and G), 1 mMM 2-mercaptoethanol (A, B, C and G), 0.1-mu-M Hg2+ (A, C and G), 0.1 mM PMSF (C and F), 1 mM TPCK (C and F), 1 mM Zn2+ (C), 60-mu-M BPP5a and 40-mu-M BPP9a (D), 0.1-mu-M phosphoramidon (E) and 3 mM sodium p-hydroxymercuribenzoate (G).6. The properties of some of these bradykinin inactivating activities correspond 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 are being purified in order to obtain a more accurate characterization.ESCOLA PAULISTA MED SCH,DEPT BIOQUIM,RUA 3 DE MAIO 100,CAIXA POSTAL 20372,BR-04044 SAO PAULO,BRAZILESCOLA PAULISTA MED SCH,DEPT MED,DISCIPLINA NEFROL,BR-04044 SAO PAULO,BRAZILESCOLA PAULISTA MED SCH,DEPT BIOQUIM,RUA 3 DE MAIO 100,CAIXA POSTAL 20372,BR-04044 SAO PAULO,BRAZILESCOLA PAULISTA MED SCH,DEPT MED,DISCIPLINA NEFROL,BR-04044 SAO PAULO,BRAZILWeb of Scienc