Partial Purification and Characterization of Rat-Liver Messenger RNA Coding for Phosphoenolpyruvate Carboxykinase (GTP)

Abstract

The mRNA coding for the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was partially purified from the liver of cyclic AMP-treated rats by a procedure involving multiple oligo(dT)-cellulose chromatographies and sucrose gradient fractionations. The purification was monitored by translational assay using a wheat germ extract. Relative to RNA bound once to oligo(dT)-cellulose, the final material was enriched 20-fold in template activity for phosphoenolpyruvate carboxykinase synthesis. With this RNA preparation, cell-free enzyme synthesis amounted to 5% of total mRNA-directed protein synthesis. The apparent sedimentation coefficient of phosphoenolpyruvate carboxykinase mRNA in sucrose gradients was between 20 and 22 S, corresponding to an average MW of 0.93 .times. 106. By formamide/polyacrylamide gel electrophoresis the MW of the enzyme mRNA was estimated at between 0.91 .times. 106 and 1.12 .times. 106. Considerable non-coding sequence(s) are probably present in the mRNA. Approximately 20% of the enzyme mRNA in rat liver failed to bind to oligo(dT)-cellulose, presumably because of the absence of a poly(A) segment. The translation of phosphoenolpyruvate carboxykinase mRNA by the wheat germ extract was inhibited in the presence of 7-methylguanosine 5''-pbosphate. The enzyme mRNA appears, therefore, to have a cap at the 5'' end.