Purification and Characterization of Two Soluble C1?-Activated Arginyl Aminopeptidases from Human Brain and Their Endopeptidase Action on Neuropeptides

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Abstract
Two closely related Cl--activated arginyl aminopeptidases (I and II) wre purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-139 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 .mu.M (I) and 37 .mu.M (II) and for Arg-AMC hydrolysis they were 167 .mu.M (I) and 125 .mu.M (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 .mu.M, respectively, with Arg-AMC substrate).