A new type of major aminopeptidase in bovine brain.
A new type of major aminopeptidase was purified from bovine brain by ammonium sulfate fractionation and TMAE-fractogel (anion exchange), arginine-Sepharose 4B, Sephadex G-150, and Sephadex G-100 column chromatography. The purified enzyme showed a maximum activity at pH 7.2, and its 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, bestatin, amastatin, actinonin, leuhistin and probestin; and very low concentrations of Cu2+, Cd2+, Pb2+, Al3+, Fe3+, and Zn2+ inhibited activity. The enzyme hydrolyzed several amino acyl-7-amido-4-methylcoumalin derivatives (amino acid-MCA). The order of MCA-substrate specificity expressed as kcat/Km is Lys-MCA > Arg-MCA > Leu-MCA > Met-MCA > Phe-MCA > Tyr-MCA > Ala-MCA >> Gly-MCA, Pro-MCA, Ser-MCA, Asn-MCA. Immunoreactivity of the antibody against the purified aminopeptidase was observed in human brain and most rat tissues examined including brain, liver, kidney, lung, heart, and skeletal muscle at the same molecular size as in bovine brain aminopeptidase. Most of the Lys-, Leu-, Met-, and Phe-MCA degrading activity in crude bovine and human brain extracts was absorbed by the aminopeptidase IgG, suggesting that this aminopeptidase is a major enzyme, sharing at least Lys-, Leu-, Met-, and Phe-MCA degrading aminopeptidase activities in the brains.
Yanagisawa
J
J
Department of Molecular Life Science, Tokai University, School of Medicine, Isehara, Kanagawa, 259-11, Japan.
Tsuda
M
M
Ohkubo
T
T
Hiyoshi
M
M
Kamiguchi
H
H
Tsukamoto
H
H
Yamamura
M
M
Kocha
T
T
Aoyagi
T
T
D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013045:Species Specificity; D013379:Substrate Specificity
D000596:Amino Acids; D007074:Immunoglobulin G; D011480:Protease Inhibitors; D000626:Aminopeptidases