Purification of ornithine carbamoyltransferase from kidney bean (Phaseolus vulgaris L.) leaves and comparison of the properties of the enzyme from canavanine-containing and -deficient plants |
| |
Authors: | Yi Lee Bang Ook Jun Sang-Gu Kim Young Myung Kwon |
| |
Affiliation: | (1) Department of Biology and Research Center for Cell Differentiation, Seoul National University, Seoul 151–742, Korea, KR;(2) Department of Biology, Kangnung University, Kangnung 210–702, Korea, KR |
| |
Abstract: | Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure, using δ-N-(phosphonoacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles citrulline per minute per milligram of protein at 35 °C and pH 8.0. The enzyme represents approximately 0.01% of the total soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, l-cysteine and S-carbamoyl-l-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline. The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines the canavanine synthetic activity of the urea cycle. Received: 22 July 1997 / Accepted: 4 December 1997 |
| |
Keywords: | : Canaline Cysteine Ornithine Ornithine carbamoyltransferase Phaseolus |
本文献已被 SpringerLink 等数据库收录! |
|