Synthesis of liver glycogen from 3-C14-DL-malate: evidence for complete isotopic equilibration of C4-dicarboxylic acids.

There are two known mechanisms of synthesis of phosphopyruvate in animal tissues. One of these is direct phosphorylation of pyruvate by adenosine 5’(pyro)-triphosphate, catalyzed by pyruvate kinase, and the other phosphorylative decarboxylation of oxaloacetate in the presence of inosine triphosphate and phosphoenolpyruvate carboxykinase. Present evidence has been interpreted as suggesting that in liver both mechanisms may be operative. This interpretation rests on data showing that, although C-l, C-2, C-5, and C-6 of the glucosyl residues of liver glycogen become especially enriched with isotope when formed from 2-C13or 2-C14-lactate or pyruvate (l-3), the isotope concentrations of C-2 and C-5 are greater than the amounts of isotope in C-l and C-6, respectively. This inequality of isotope concentrations has been explained by assuming that glucosyl residues labeled in positions 2,5 (direct phosphorylation of pyruvate) are mixed with others labeled in positions 1,2,5, and 6 (phosphorylative decarboxylation of oxaloacetate) . This interpretation of the data presupposes that oxaloacetate, labeled equally in positions 2 and 3, is an intermediate in the reaction, and that the isotope is distributed in this way because of the presence in the pathway of a symmetrical C4-dicarboxylic acid intermediate. The C4-dicarboxylic acid pathway of formation of 2,3-C14phosphopyruvate from 2-C14-pyruvate is illustrated in Fig. 1. It will be seen (Pathway 1) that if the rate of isotopic equilibration of malate and fumarate is more rapid than the rate of oxidation of 2-CWmalate, the specific activities of C-2 and C-3 of malate become equal and 2,3-C14-phosphopyruvate is the sole product of the reaction. On the other hand, if oxidation of a significant proportion of 2-C14-malate takes place before equilibration of Cl4 in malate (Pathway b), 2-C14-phosphopyruvate would be formed. Thus it is conceivable that the higher specific activities of C-2 and C-5 than of C-l and C-6, respectively, reflect incompleteness of isotopic equilibration of malate. The present studies were conducted for the purpose of assessing the extent of isotopic equilibration of positions 2 and 3 of malate in rat liver. For this purpose 3-C14-on-malate was synthesized and administered to fasting rats. Liver glycogen was isolated and subjected to degradative procedures in order to measure the specific activities of C-4, C-5, and C-6 in the glucosyl residues. The experiment revealed that the specific activities of C-5 and C-6 were equal, indicating that isotopic equilibration of C-2 and C-3 in malate occurred more rapidly than oxidation of the malate.