The inhibition of phosphoenolpyruvate carboxykinase following in vivo chronic phenobarbital treatment in the rat is due to a post-translational event.
Chronic treatment of rats with phenobarbital has been reported to decrease gluconeogenesis in rat hepatocytes by a 50% inhibition of phosphoenolpyruvate (P-pyruvate) carboxykinase activity [Argaud, D., Halimi, S., Catelloni, F. & Leverve, X. (1991) Biochem. J. 280, 663-669]. Contrary to the current knowledge of P-pyruvate carboxykinase regulation, we failed to find a diminution of either P-pyruvate carboxykinase protein (by using a polyclonal antibody) or P-pyruvate carboxykinase mRNA, in the liver of rats treated with phenobarbital for 2 weeks. Kinetic studies of P-pyruvate carboxykinase activity, measured by either carboxylation of P-pyruvate or decarboxylation of oxaloacetate, revealed a decrease in both V(max) and Km after phenobarbital treatment, whereas the nutritional state affected only the V(max), as expected. Assessment of P-pyruvate carboxykinase specificity was confirmed by the full inhibition of the enzyme with its specific inhibitor 3-mercaptopicolinate in the micromolar range. P-Pyruvate carboxykinase, purified either by ammonium sulfate fractionation or by immunoprecipitation, exhibited a similar decrease in affinity after phenobarbital treatment. Although the molecular mass does not appear to be altered, the pH sensitivity to 3-mercaptopicolinate inhibition and the enzyme recovery after immunoprecipitation both seemed to be affected. This leads us to propose that the effect of chronic phenobarbital treatment on P-pyruvate carboxykinase activity is not the result of transcriptional regulation but is exerted at the post-translational level.
Chauvin
C
C
Laboratoire de Bioénergétique Fondamentale et Appliquée, Université Joseph Fourier, Grenoble, France.
Brilloit-Petit
C
C
Argaud
D
D
Catelloni
F
F
Velours
J
J
Leverve
X M
XM
D000824:Animal Nutritional Physiological Phenomena; D000818:Animals; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar
D012333:RNA, Messenger; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D010634:Phenobarbital