Fate of Isotopic Carbon in Kidney Mitochondria Synthesizing Precursors for Glucose from Pyruvate and Bicarbonate

Abstract

Abstract Rat kidney mitochondria in the presence of ATP, orthophosphate, and magnesium ions convert pyruvate and bicarbonate primarily to malate, fumarate, and citrate. Pyruvate carboxylase activity was estimated by measuring the incorporation of radioactive bicarbonate into tricarboxylic acid cycle intermediates. In the presence of added α-ketoglutarate or glutamate, the incorporation of 14CO2 into organic acids was greatly increased. The addition of both glutamate and α-ketoglutarate, while greatly increasing 14CO2 incorporation, led to the inhibition of aspartate formation. The low specific radioactivity of malate and citrate obtained from carboxylation of pyruvate with radioactive bicarbonate indicates that some of the radioactive bicarbonate is again lost during metabolism of the tricarboxylic acid cycle and suggests that the unlabeled CO2, derived from oxidative decarboxylation of pyruvate, may be used preferentially for carboxylation. When pyruvate-1-14C instead of radioactive bicarbonate was labeled, the specific radioactivity of isolated malate and citrate was considerably higher. On the basis of these findings, pyruvate carboxylase activity is significantly higher than that previously reported in the literature based on the incorporation of radioactive bicarbonate into organic acids. Oxalacetate also exerts a regulatory effect on carboxylation of pyruvate as evidenced by the large increase in the 14CO2 incorporation in the presence of added glutamate.