Characterization of bovine liver ornithine aminotransferase

Abstract

소의 간으로부터 ornithine aminotransferase를 ammonium-sulfate침전, DEAE-cellulose chromatography, DEAE-Sepha-dex chromatography, Sephadex G-200 gel filtration chromatography를 이용하여 6.4%의 회수율로 209배 정제하였다. 이 정제된 ornithine aminotransferase를 전기이동법으로 조사한 결과 하나의 단백질 띠로 나타났으며 그 정제도가 높음을 확인할 수 있었다. Gel filtration법으로 효소의 분자량이 88,000으로 추정되었고, SDS-polyacrylamide gel electrophoresis를 행하여 분자량이 42,000인 두개의 subunit로 구성되었음을 알 수 있었다. 이 효소의 isoelectric focusing법으로 측정된 pI는 5.33이었으며, pH적정점은 8.0이었다. 또한 효소 한 분자당 pyridoxal phosphate 한 분자가 결합되었으며, 효소 한 분자당 존재하는 thiol group은 아홉개이였다. 이 효소의 L-ornithine과 α-ketoglutate에 대한 Km은 각각 5.78mM과 0.5mM이었고, L-valie은 L-ornithine에 대하여 competitive inhibitor이었으며 그 Ki는 9.5mM이었다. ; Bovine liver ornithine aminotransferase ( L-ornithine: 2-ox-oacid aminotransferase, EC 2.6.1.13) was purified as high as 209 fold (6.4% yield)and its molecular weight was estimated to be 88,000 by the method of gel filtration. Two dimensional combined isoelectric focusing-SDS polyacrylamide gel electrophoresis revealed a single band with an apparent molecular weight of 42,000, suggesting that the enzyme consists of two subunits. pI of the enzyme was 5.33 and its optimum pH was found to be 8.0. The enzyme contained approximately one mole of pyridoxal phosphate per mole and the total number of thiol groups per mole was estimated to be nine. Km s of the enzyme for L-ornithine and α-ketoglutate were determined to be 5.78mM and 0.5mM respectively. L-valine was competitive with L-ornithine and its Ki was found to be 9.5mM.