The regulation of phosphoenolpyruvate synthesis in pigeon liver

Abstract

The intracellular location and maximal activities of enzymes involved in phosphoenolpyruvate synthesis have been investigated in pigeon liver. Enolase and pyruvate kinase were cytoplasmic, and the activites were 50-60 and 180-210 [mu]moles/min./g dry wt. at 25[degree] respectively. Phosphoenolpyruvate carboxykinase was present exclusively, and nucleoside diphosphokinase predominantly, in the mitochondria; the particles had to be disrupted to elicit maximal activities, which were 27-33 and 400-600 [mu]moles/ min./g dry wt. at 25[degree] respectively. The activities of all four enzymes did not change significantly during 48 hr. of starvation. Conditions for incubation of washed isolated mitochondria were established, to give high rates of synthesis of phosphoenolpyruvate, linear with time and proportional to mitochondrial concentration. Inorganic phosphate and added adenine nucleotides were stimulatory, whereas added Mg2+ inhibited, partly owing to activation of contaminant pyruvate kinase. Phosphoenolpyruvate formation occurred from oxaloacetate, malate, fumarate, succinate, [alpha]-oxoglutarate and citrate, in decreasing order of effectiveness. The steady-state ATP/ADP ratio of mitochondrial suspensions was decreased in the presence of added 2.5 mM-Mg2+ (owing to stimulation of adenylate kinase and possibly of an adenosine triphosphatase), 0.5 mM-Ca2+ or 0.04 mM-dinitrophenol. In each case the rate of substrate removal and oxygen uptake was increased, whereas phosphoenolpyruvate synthesis was inhibited. Citrate formation was enhanced, owing to de-inhibition of citrate synthase. These effects were not primarily related to changes in the oxaloacetate concentration. Both phosphoenolpyruvate carboxykinase and nucleoside diphosphokinase were active within the atractyloside-sensitive barrier to the mitochondrial metabolism of added adenine nucleotides. There was no correlation between the rate of substrate-level phosphorylation associated with the oxidation of [alpha] -oxoglutarate, and the synthesis of phosphoenolpyruvate. The results suggest that phosphoenolpyruvate formation in pigeon-liver mitochondria is regulated partly by the phosphorylation state of the adenine and guanine nucleotides, and partly by variations in the oxaloacetate concentration, all in the mitochondrial matrix. Phosphoenolpyruvate is assumed to be the metabolite transported from the mitochondria to the cytoplasm during gluconeogenesis from oxaloacetate in pigeon liver.