Purification and characterization of ornithine aminotransferase from chicken liver

Abstract

닭 간을 균질화하여 cell fractionation하였더니 ornithine aminotransferase가 주로 mitochondrial fraction에 존재하였다. 이 효소를 열처리, ammonium sulfate fractionation, DEAE-cellulose ion exchange chromatography, Sephadex G-200 filtration, CM-Sephadex column chromatography 의 순으로 약 298배 정제해서 얻은 효소액의 specific activity는 566 x 10^-3 units/mg protein이었으며 disc plyyacrylamide gel electrophoresis를 시행한 결과 하나의 band가 얻어졌다. 이 정제된 효소를 이용해서 몇가지 성질을 조사해본 결과는 다음과 같다. SDS-polyacrylamide gel electrophoresis에 의해 측정된 분자량은 62,000이었고 gel filtration에 의한 값은 63,000이었으므로 이 효소는 monomer로 존재한다고 생각되었다. L-or-nithine과 α-ketoglutarate에 대한 Km값은 각각 6.31mM, 1.03mM이었으며 이 효소는 기질의 농도가 높을 때 저해되었고, 또한 3mM p-chloromercuriphenylsulfonic acid를 가해주면 80% 저해되었다. Glyoxylate, oxaloacetate, pyruvate도 α-ketoglutarate보다는 못하지만 amino group acceptor로서 작용하였으며 효소활성에 대한 최적 pH는 7.3이었다. ; Ornithine aminotransferase (EC 2.6.1.13., OAT) was purified from chicken liver and several properties of the enzyme were studied. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated as 62,000 by SDS-polyacrylamide gel electrophoresis, and as 63,000 by gel filtration. The Michaelis constants for ornithine and α-ketoglutarate were found to be 6.31mM and 1.03mM, respectively. The enzyme was inhibited either by high substrate concentrations or by p-chloromercuriphenylsulfonic acid. Glyoxylate, oxaloacetate and pyruvate could act as amino group acceptors, but they were much less effective than α-ketoglutarate. Optimum pH was found to be 7.3.