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.
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 delta-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 degrees 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.
Lee
Y
Y
Department of Biology, Seoul National University, Korea.
Jun
B O
BO
Kim
S G
SG
Kwon
Y M
YM
D000818:Animals; D002172:Canavanine / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008970:Molecular Weight; D009952:Ornithine / Q000378:metabolism; D009954:Ornithine Carbamoyltransferase / Q000302:isolation & purification* / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D010946:Plants, Medicinal; D011817:Rabbits; D013379:Substrate Specificity
D002172:Canavanine; D002221:Carbamyl Phosphate; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase