Ornithine Carbamoyltransferase from Spinacea oleracea: Purification and Characterization |
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Authors: | E Bellocco C Di Salvo G Laganà A Galtieri S Ficarra A Kotyk U Leuzzi |
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Institution: | (1) Department of Organic and Biological Chemistry, University of Messina, Salita Sperone 31, Villaggio S. Agata, I-98166 Messina, Italy;(2) Institute of Physiology, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic |
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Abstract: | Ornithine carbamoyltransferase (OCT) from spinach (Spinacea oleracea L.) was purified to homogeneity and studied for some kinetic and structural properties. The enzyme showed a specific activity of 436 U mg–1, its molecular mass was approximately 118 kDa as estimated by Sephacryl S-200 gel filtration chromatography, the purified protein ran as a single band of 38 kDa in sodium dodecyl sulfate-polyacryamide gel electrophoresis. The enzyme catalyses an ordered bi-bi-sequential reaction in which carbamoyl phosphate binds first, followed by L-ornithine; L-citrulline leaves first, followed by phosphate. The Michaelis constant was 0.19 mM for L-ornithine and 13.1 µM for carbamoyl phosphate; the dissociation constant for the enzyme and carbamoyl phosphate complex was of 19 µM. The Km of the reaction decreases from pH 6.0 to pH 10.4. The enzyme is heat-labile, but it was protected from thermal inactivation by substrates; more by ornithine alone than by two substrates acting together. |
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Keywords: | enzyme kinetics enzyme purification -N-(phosphonoacetyl)-L-ornithine" target="_blank">gif" alt="delta" align="BASELINE" BORDER="0">-N-(phosphonoacetyl)-L-ornithine spinach |
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