Purification and characterization of ornithine carbamoyltransferase from the chloroplasts of Canavalia lineata leaves

• Published in: Plant science (Limerick) Vol. 122; no. 2; pp. 217 - 224
• Main Authors: Lee, Yi, Lee, Chin Bum, Kim, Sang-Gu, Kwon, Young Myung
• Format: Journal Article
• Language: English
• Published: Shannon Elsevier Ireland Ltd 28.02.1997; Elsevier Science
• Subjects: Biological and medical sciences; Canaline; Canavalia lineata; Chloroplasts; Enzymes; Fundamental and applied biological sciences. Psychology; Metabolism; Ornithine; Plant physiology and development; OCT; Canavalia lineata; Chloroplasts; Ornithine; Canaline; CP; Trimer; Purification; Enzyme; Transferases; Plant leaf; Ornithine carbamoyltransferase; Characterization; Leguminosae; Enzymatic activity; Dicotyledones; Substrate specificity; Angiospermae; Spermatophyta; Kinetics; Chloroplast
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/S0168-9452(96)04547-5

Ornithine carbamoyltransferase (OCT) was characterized from the chloroplasts of Canavalia lineata leaves. The OCT was purified 419-fold with a yield of 7.9% by streptomycin sulfate precipitation, ammonium sulfate fractionation, Sephacryl S-300 gel filtration, hydroxylapatite, and reactive red dye chromatographies. The approximate molecular weight of OCT was 107 kD by measurement from Sephacryl S-300 gel filtration. The subunit molecular weight of the enzyme was 38 kD based on SDS-PAGE. These results suggest that the native enzyme is a trimer. The OCT of C. lineata used not only ornithine but also canaline as a substrate. As for the ornithine-dependent activity, the enzyme had a pH optimum at 8.5 and Michaelis constants of 2.4 mM for l-ornithine and 0.21 mM for carbamoyl phosphate (CP). As for the canaline-dependent activity, Michaelis constants for l-canaline and CP were 11 and 0.13 mM, respectively, at pH 8.0 (optimum pH of canaline-dependent activity). S-carbamoyl- l-cysteine and l-cysteine were very strong inhibitors for the enzyme activity. Inhibition by l-cysteine was shown to be competitive with respect to ornithine, but that was shown to be noncompetitive with respect to canaline. The ornithine-dependent activity was inhibited by 90% in the presence of 2 mM Cu 2+ and the canaline-dependent activity was inhibited by 91 and 100% with 2 mM Cd 2+ and 2 mM Hg 2+, respectively.