Purification and characterization of two soluble Cl(-)-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides.
Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were 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, adrenocorticotropin18-39 (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 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).
McDermott
J R
JR
MRC Neuroendocrinology Unit, Newcastle General Hospital, Newcastle upon Tyne, England.
Mantle
D
D
Lauffart
B
B
Gibson
A M
AM
Biggins
J A
JA
D000368:Aged; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000838:Anions; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D002412:Cations; D002540:Cerebral Cortex / Q000201:enzymology*; D002712:Chlorides / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D003374:Coumarins / Q000378:metabolism; D004355:Drug Stability; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009479:Neuropeptides / Q000378:metabolism*; D013379:Substrate Specificity
D000838:Anions; D002412:Cations; D002712:Chlorides; D003374:Coumarins; D009479:Neuropeptides; C041841:arginine 4-methyl-7-coumarylamide; D001120:Arginine; D010450:Endopeptidases; D000626:Aminopeptidases; C019721:aminopeptidase B