Abstract
The administration of N6, O2'-dibutyryl cyclic AMP and theophylline to fasted-refed rats produces an 8-fold stimulation of the relative rate of hepatic phosphoenolpyruvate carboxykinase synthesis in 90 min, as measured by isotopic immunochemical techniques in vivo. The mechanism of this induction was studied first by using a homologous, noninitiating cell-free protein-synthesizing system derived from the liver of fasted-refed, cyclic AMP-treated rats. In such a system, a 5-fold increase in phosphoenolpyruvate carboxykinase synthseis is observed at 20 min post-treatment and a 9-fold stimulation at 75 min, indicating a rapid increase in the number of ribosomes engaged in the translation of the enzyme mRNA after exposure to cyclic AMP. The level of functional mRNA coding for phosphoenolpyruvate carboxykinase was then assayed in a wheat germ protein-synthesizing system capable of using rat liver mRNA as template. The template activity for phosphoenolpyruvate carboxykinase synthesis is greatly increased in the poly(A)-containing RNA isolated from cyclic AMP-induced animals. Both the increase in the capacity of the liver extract for in vitro phosphoenolpyruvate carboxykinase synthesis and the emergence of enzyme mRNA detected in the wheat germ assay are completely prevented by a pretreatment with cordycepin at doses which inhibit the appearance in the cytoplasm of newly synthesized poly(A)-containing RNA. These data demonstrate that the induction of hepatic phosphoenolpyruvate carboxykinase by cyclic AMP is characterized by the rapid build-up of newly synthesized, actively translated mRNA coding for the enzyme. The messenger accumulation could be due to an increase in the rate of its production or a decrease in the rate of its degradation.
Highlights
These data demonstrate that the induction of hepatic phosphoenolpyruvate carboxykinase by cyclic AMP is characterized by the rapid build-up of newly synthesized, actively translated mRNA coding for the enzyme
This consistency constitutes a decisive advantage of the wheat germ extract over the rabbit reticulocyte lysate, whose capability to carry out P-enolpyruvate carboxykinase synthesis was shown in a previous paper [41]
We report that the effect of dibutyryl cyclic AMP
Summary
’ Abbreviations phoenolpyruvate RNA containing without polyadenylic azine-N’-2.ethanesulfonic tyryl cyclic AMP, used are: P-enolpyruvate carboxykinase, phoscarboxykinase (GTP) (EC 4.1.1.32); Poly(A)+RNA, a polyadenylic acid sequence; Poly(A)-RNA, RNA acid sequence; Hepes, N-2-hydroxyethylpiperacid. A sample of incubation mixture was taken for the determination of total hot t,richloroacetic acid-precipitable radioactivity and the rest was centrifuged at 164,000 x g,,, for 60 min at 4”. HCl, pH 7.6, 103 rn~ potassium acetate, 24 IIIM KCl, 1.4 mM magnesium (acetate),, 2.3 IIIM dithiothreitol, 1.9 rn~ 2.mercaptoethanol, m&r ATP, 0.1 IIIM GTP, 8 rn~ phosphocreatine, 40 @g of creatine phosphokinaseiml, 0.1 IIIM spermine, 30 PM 19 amino acids, 40 &i of [“Hlleucineiml, and appropriate poly(A)+RNA concentrations in a total volume of 0.59 ml containing. Carboxykinase; Determination of Radioactivity in Enzyme and in Total Protein -After cellfree protein synthesis using the liver S-17 extract, a volume of high speed supernatant containing. The precipitation of total protein from the incubation mixtures and the estimation of their radioactivity were performed as described by Ballard et al [22] in the presence of carrier bovine serum albumin
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