Kinin and enkephalin conversion by an endothelial, plasma membrane carboxypeptidase

Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was determined that a vascular plasma membrane preparation contains a carboxypeptidase capable of converting kinins (B2 agonists) to des(Arg)kinins (B1 agonists) by hydrolysis of C-terminal Arg. The plasma membrane carboxypeptidase also converted Leu5-enkephalin-Arg6 to Leu5-enkephalin. Carboxypeptidase activity was significantly higher in cultured endothelial (1.47 +/- 0.4 units/mg) than in cultured smooth muscle cells (0.16 +/- 0.4 units/mg). Both the vascular and endothelial activities had neutral pH optima and were activated 4- to 5-fold by 0.1 mM CoCl2. The carboxypeptidase N inhibitor MERGETPA (D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid) inhibited the plasma membrane bound carboxypeptidase with an I50 of 0.3 microM. Conversion was also inhibited by o-phenanthroline and EDTA, whereas inhibitors of aminopeptidases (bestatin, puromycin), endopeptidases (phosphoramidon), "enkephalinase" (ZINCOV) or enkephalin convertase (PCMS) were without effect. The affinity of the endothelial plasma membrane carboxypeptidase for bradykinin (Km = 56.8 +/- 4.7 microM) was higher than that for Leu5-enkephalin-Arg6 (Km = 92.7 +/- 10.1 microM), whereas the maximal rates of conversion (calculated per mg of endothelial plasma membrane protein) were similar (17.1 and 21.3 nmoles/min/mg respectively). These results demonstrate that a carboxypeptidase is present on the cell surface of vascular endothelium which can convert kinins and enkephalins in the micro-environments of vascular cell surface receptors.