Effects of dimethyl sulfoxide and glycerol on catalytic and regulatory properties of glutamine-dependent carbamoyl phosphate synthase from rat liver and dual effects of uridine triphosphate. |
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Authors: | H Ishida M Mori M Tatibana |
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Institution: | Department of Biochemistry, Chiba University School of Medicine, Inohana, Chiba 280, Japan |
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Abstract: | The cryoprotectants dimethyl sulfoxide and glycerol markedly affected the activity of glutamine-dependent carbamoyl phosphate synthase (EC 2.7.2.9) of rat liver, the first enzyme of de novo pyrimidine biosynthesis. The apparent Km for MgATP2? decreased with increases in the solvent concentrations, from 7.0 mm without the solvents to 0.1 and 0.8 mm in the presence of 25% () dimethyl sulfoxide and 30% () glycerol, respectively. The apparent Km for bicarbonate also decreased with these solvents, while that for glutamine was not significantly affected. The response in the maximal velocity to the solvents was biphasic; the value of V increased 1.39- and 1.18-fold with 7.5% dimethyl sulfoxide and 10% glycerol, respectively, but then decreased as the concentrations of the solvents were further increased. The extents of inhibition by 25% dimethyl sulfoxide and 30% glycerol were 63 and 44%, respectively. The effects of 5-phosphoribosyl 1-pyrophosphate and MgUTP, allosteric effectors, were greatly modified by these solvents. Kinetic parameters as affected by the effectors in the presence of various concentrations of the solvents are described. A notable observation was that MgUTP, a potent inhibitor under ordinary conditions, stimulated the activity in the presence of high concentrations of dimethyl sulfoxide; in the presence of 30% dimethyl sulfoxide and 1 mm MgATP2?, 0.5 to 2.0 mm MgUTP enhanced the enzymatic activity about twofold. MgUTP at higher concentrations was inhibitory. The dimethyl sulfoxide-dependent dual effects of MgUTP indicate the possible existence of at least two MgUTP-binding sites on the enzyme molecule. |
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