Messenger RNA for renal phosphoenolpyruvate carboxykinase (GTP). Its translation in a heterologous cell-free system and its regulation by glucocorticoids and by changes in acid-base balance.

ABSTRACT

A translational assay was used to measure the level of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in the rat kidney in various conditions in which the enzyme is induced. RNA extracted from whole kidneys was chromatographed on oligo(dT)-cellulose to select poly(A)-containing RNA. This crude mRNA preparation was able to stimulate amino acid incorporation into protein in a cell-free system containing an extract of wheat germ. Phosphoenolpyruvate carboxykinase could be detected among the polypeptides synthesized and quantitated by immunoprecipitation with a monospecific antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of enzyme synthesized was proportional to the quantity of RNA added. The level of mRNA coding for phosphoenolpyruvate carboxykinase is increased 3-fold 6 h after triamcinolone injection. Translatable enzyme mRNA also increases 3-fold within 6 h of the onset of metabolic acidosis caused by an ammonium chloride load. In both cases, the increase in functional mRNA is commensurate with the stimulation of enzyme synthesis measured in vivo. Glucocorticoid administration and acidosis cause additive increases in the level of translatable phosphoenolpyruvate carboxykinase mRNA. The inductive effect of acidosis is preserved in the absence of the adrenals, hypophysis, thyroid, and parathyroids.