INHIBITION AND METAL-ION ACTIVATION OF PIG-KIDNEY AMINOPEPTIDASE-P - DEPENDENCE ON NATURE OF SUBSTRATE

Pig kidney aminopeptidase P (AP-P; EC 3.4.11.9) has been purified to h omogeneity after its solubilisation from brush border membranes by pho sphatidylinositol-specific phospholipase C. The effects of various act ivators and inhibitors of AP-P activity have been examined with a numb er of different substrates for the enzyme. The hydrolysis of bradykini n and ArgProPrO is inhibited at Mn2+ concentrations above 10(-5) M, wh ereas the hydrolysis of other substrates (GlyProHyp, beta-casomorphin, substance P) is substantially activated, with 4-10 mM Mn2+ being opti mal. The thiol reagent, p-chloromercuriphenylsulphonic acid, inhibits the hydrolysis of GlyProHyp but markedly activates the hydrolysis of b radykinin. A number of inhibitors of angiotensin converting enzyme (AC E; EC 3.4.15.1), previously reported to inhibit the hydrolysis of GlyP roHyp, have no effect on the hydrolysis of bradykinin except in the pr esence of Mn2+. Differences were also observed in the degree of inhibi tion of GlyProHyp and bradykinin hydrolysis by EDTA and their reactiva tion by divalent cations. The hydrolysis of GlyProHyp follows Michaeli s-Menten kinetics with a K-m value of 2.7 mM. Bradykinin inhibits GlyP roHyp hydrolysis with an I-50 Of 1.4 mu M. The hydrolysis of bradykini n by AP-P reveals anomalous nonlinear kinetics indicative of negative cooperativity or the presence of more than one active site for this su bstrate. These results indicate that substrates for AP-P can be divide d into 2 groups based on their responses to inhibitors and cation acti vators.