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PMID:1148171

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Citation

Mori, M, Ishida, H and Tatibana, M (1975) Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver. Biochemistry 14:2622-30

Abstract

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.

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Keywords

Amidohydrolases/isolation & purification; Amidohydrolases/metabolism; Animals; Aspartate Carbamoyltransferase/isolation & purification; Aspartate Carbamoyltransferase/metabolism; Binding Sites; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/isolation & purification; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism; Centrifugation, Density Gradient; Chromatography; Dimethyl Sulfoxide; Glycerol; Hydroxyapatites; Kinetics; Liver/metabolism; Mice; Multienzyme Complexes/isolation & purification; Multienzyme Complexes/metabolism; Orotic Acid; Protein Binding; Pyrimidines/biosynthesis; Rats; Spleen/enzymology

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