A NEW-TYPE OF MAJOR AMINOPEPTIDASE IN BOVINE BRAIN

A new type of major aminopeptidase was purified from bovine brain by a mmonium sulfate fractionation and TMAE-fractogel (anion exchange), arg inine-Sepharose 4B, Sephadex G-150, and Sephadex G-100 column chromato graphy. The purified enzyme showed a maximum activity at pH 7.2, and i ts molecular size was estimated to be 98,000 by gel filtration and 104 ,000 by SDS-PAGE with or without 2-mercaptoethanol. Further properties were activation by thiol reagents; inhibition by EDTA, puromycin, bes tatin, amastatin, actinonin, leuhistin and probestin; and very low con centrations of Cu2+, Cd2+, Pb2+, Al3+, Fe3+, and Zn2+ inhibited activi ty. The enzyme hydrolyzed several amino acyl-7-amido-4-methylcoumalin derivatives (amino acid-MCA). The order of MCA-substrate specificity e xpressed as kcat/K-m is Lys-MCA > Arg-MCA > Leu-MCA > Met-MCA > Phe-MC A > Tyr-MCA > Ala-MCA much greater than Gly-MCA, Pro-MCA, Ser-MCA, Asn -MCA Immunoreactivity of the antibody against the purified aminopeptid ase was observed in human brain and most rat tissues examined includin g brain, liver, kidney, lung, heart, and skeletal muscle at the same m olecular size as in bovine brain aminopeptidase. Most of the Lys-, Leu -, Met-, and Phe-MCA degrading activity in crude bovine and human brai n extracts was absorbed by the aminopeptidase IgG, suggesting that thi s aminopeptidase is a major enzyme, sharing at least Lys-, Leu-, Met-, and Phe-MCA degrading aminopeptidase activities in the brains. (C) 19 96 Academic Press, Inc.