MAJOR KININASES IN RAT URINE ARE NEUTRAL ENDOPEPTIDASE AND CARBOXYPEPTIDASE Y-LIKE EXOPEPTIDASE

Two types of kininases were separated from rat urine by gel-filtration on a column of Superdex 200 and characterized as a membrane-bound neutral endopeptidase (EC 3.4.24.11) and a carboxypeptidase Y-like exopeptidase. The neutral endopeptidase which was found in a void volume fraction, liberated first Phe8-Arg9 and then Phe5-Ser6-Pro7 from bradykinin (BK)*. The kininase activity was inhibited by phosphoramidon and thiorphan but not by captopril. Another kininase with an apparent molecular weight of 93,000 cleaved rapidly BK into BK1-8 (des Arg9 BK) and BK1-7 (des Phe8-Arg9 BK) into BK1-6 (des Pro7-Phe8-Arg9 BK). The kininase activity was inhibited by inhibitors for serine proteases and cysteine proteases and by metal ions (Co2+, Hg2+ or Ag+), but not by inhibitors for metal proteases. The kininase activity was also inhibited by a rabbit antipeptide IgG which was raised against a synthetic peptide with the amino acid sequence of human and mouse carboxypeptidase Y-like peptidases. These results indicate that the exopeptidase is not carboxypeptidases N or M but a member of a family of human and mouse carboxypeptidase Y-like exopeptidases which are identified as protective proteins associated with lysosomal beta-galactosidase and neuraminidase or platelet peptidase with deamidase activity. When rat urine was incubated with BK, the generation of either BK1-8 or BK1-7 was inhibited in the presence of either DFP* or phosphoramidon. In the presence of both of DFP and phosphoramidon, degradation of BK was inhibited, whereas captopril had no effect on the degradation of BK. These results demonstrate that major kininases in rat urine are neutral endopeptidase and carboxypeptidase Y-like exopeptidase.