I. Fox, W. Kelley
Sep 25, 1971
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Influential Citations
159
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Journal
The Journal of biological chemistry
Abstract
Abstract Phosphoribosylpyrophosphate (PP-ribose-P) synthetase was found to be distributed in all human tissues assayed and has been purified 5100-fold from human erythrocytes. It has an absolute requirement for inorganic phosphate. Magnesium was the most effective divalent cation at both high and low concentrations. No requirement for potassium was found. The pH optimum was 7.2 to 7.4. The isoelectric point was 4.7 ± 0.2. With the usual substrates, ATP and ribose-5-P, the reaction produces equimolar amounts of PP-ribose-P and AMP. Of a number of purine and pyrimidine nucleotides tested as possible substrates, the purified enzyme was active only with ATP and dATP. Ribulose-5-P was 34% as effective a substrate as ribose-5-P, while the enzyme had virtually no activity with other phosphorylated sugars. The Km values for ATP and ribose-5-P were 14 µm and 33 µm, respectively. Crude preparations of the enzyme were stabilized by dithiothreitol while the 5100-fold purified enzyme was inactivated by this compound. The enzyme was stabilized at -70°, 4°, and 60° by saturating concentrations of ATP and magnesium. Phosphate was absolutely required to maintain activity. The reversible association and dissociation of PP-ribose-P synthetase is described. The smallest native form of the enzyme has a molecular weight of 60,000 as estimated from Stokes radius and sedimentation velocity. In the presence of saturating concentrations of ATP and magnesium chloride, the enzyme consistently associates into two heavy forms estimated to have molecular weights of about 1,200,000 and 720,000. Intermediate molecular forms were observed in the presence of ATP or magnesium chloride alone. Polyacrylamide electrophoresis of the enzyme in sodium dodecyl sulfate revealed a single band with a molecular weight of 34,500. It is probable that the aggregated form is the active enzyme and that the enzyme is normally aggregated in the cell. Correlation of this reversible association and dissociation with cooperativity have not yet been documented.