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Isolation of rat liver CTP: phosphocholine cytidylyltransferase and regulation of hepatic phosphatidylcholine biosynthesis Yao, Zemin


Two kinds of affinity chromatography, CDP-choline- and CTP-Sepharose 4B, were investigated for purification of the cytosolic CTP:phosphocholine cytidylyltransferase from rat liver. The enzyme did not show strong affinity for the CDP-choline Sepharose resin, but bound to the CTP-Sepharose column in the presence of 14 mM magnesium acetate. The combination of CTP affinity chromatography with ion-exchange techniques provided about 70-fold purification of the cytosolic enzyme with a specific activity of about 90 units per milligram protein. The influence of diphenylsulfone compounds on the synthesis of phosphatidylcholine by the CDP-choline pathway was examined in isolated rat hepatocytes and HeLa cells. The administration of the sulfones (100 ug/ml), except dapsone, to HeLa cells inhibited the total [methyl-³H]choline incorporation into the cells, but did not change the rate of conversion of choline to phosphatidylcholine. The addition of the sulfones (100 ug/ml) to rat hepatocytes did not inhibit the biosynthesis of phosphatidylcholine and choline metabolism. The effect of vasopressin on the distribution of cytidylyl-transferase between cytosol and microsomes in rat hepatocytes was also investigated. The digitonin-mediated release of cytosolic cytidylyltransferase was reduced from the cells treated with vasopressin (5-20 nM) , while the enhanced rate of incorporation of [methyl-³H]choline into phosphatidylcholine was not observed.

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