Lloyd G S, Hryszko J, Hooper N M, Turner A J
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochem Pharmacol. 1996 Jul 26;52(2):229-36. doi: 10.1016/0006-2952(96)00180-3.
Pig kidney aminopeptidase P (AP-P; EC 3.4.11.9) has been purified to homogeneity after its solubilisation from brush border membranes by phosphatidylinositol-specific phospholipase C. The effects of various activators and inhibitors of AP-P activity have been examined with a number of different substrates for the enzyme. The hydrolysis of bradykinin and ArgProPro is inhibited at Mn2+ concentrations above 10(-5) M, whereas the hydrolysis of other substrates (GlyProHyp, beta-casomorphin, substance P) is substantially activated, with 4-10 mM Mn2+ being optimal. The thiol reagent, p-chloromercuriphenylsulphonic acid, inhibits the hydrolysis of GlyProHyp but markedly activates the hydrolysis of bradykinin. A number of inhibitors of angiotensin converting enzyme (ACE; EC 3.4.15.1), previously reported to inhibit the hydrolysis of GlyProHyp, have no effect on the hydrolysis of bradykinin except in the presence of Mn2+. Differences were also observed in the degree of inhibition of GlyProHyp and bradykinin hydrolysis by EDTA and their reactivation by divalent cations. The hydrolysis of GlyProHyp follows Michaelis-Menten kinetics with a Km value of 2.7 mM. Bradykinin inhibits GlyProHyp hydrolysis with an I50 of 1.4 microM. The hydrolysis of bradykinin by AP-P reveals anomalous nonlinear kinetics indicative of negative cooperativity or the presence of more than one active site for this substrate. These results indicate that substrates for AP-P can be divided into 2 groups based on their responses to inhibitors and cation activators.
猪肾氨肽酶P（AP-P；EC 3.4.11.9）经磷脂酰肌醇特异性磷脂酶C从刷状缘膜中溶解后已被纯化至同质。已用该酶的多种不同底物研究了各种AP-P活性激活剂和抑制剂的作用。当Mn2+浓度高于10(-5) M时，缓激肽和ArgProPro的水解受到抑制，而其他底物（甘氨酰脯氨酰羟脯氨酸、β-酪蛋白吗啡、P物质）的水解则被显著激活，4-10 mM Mn2+为最佳浓度。巯基试剂对氯汞苯磺酸抑制甘氨酰脯氨酰羟脯氨酸的水解，但显著激活缓激肽的水解。先前报道的一些血管紧张素转换酶（ACE；EC 3.4.15.1）抑制剂可抑制甘氨酰脯氨酰羟脯氨酸的水解，但除在Mn2+存在的情况下外，对缓激肽的水解无影响。在EDTA对甘氨酰脯氨酰羟脯氨酸和缓激肽水解的抑制程度及其被二价阳离子重新激活方面也观察到差异。甘氨酰脯氨酰羟脯氨酸的水解遵循米氏动力学，Km值为2.7 mM。缓激肽以1.4 microM的I50抑制甘氨酰脯氨酰羟脯氨酸的水解。AP-P对缓激肽的水解显示出异常的非线性动力学，表明存在负协同性或该底物有多个活性位点。这些结果表明，基于对抑制剂和阳离子激活剂的反应，AP-P的底物可分为两组。
