Oligodeoxyribonucleotide primers for calf thymus polymerase.

tion as measured by the hydroxamate reaction did not change appreciably, and furthermore, the amount of oxaloacetate synthesized was almost twice the amount of acetyl-CoA added. In other experiments enzymatic measurements of acetyl-CoA concentration gave similar results. The indirect role of acetyl-CoA is further confirmed by experiments shown in Table III. Oxaloacetate was formed from various Cl4 precursors, isolated by Celite chromatography, and degraded with AP+ to yield the fl-COOH and pyruvate. The pyruvate was isolated by ion exchange chromatography. CY402 contributes only the @-COOH, pyruvate-land -2-C’* enter only the pyruvate moiety, whereas acetyll-CY4-CoA labels neither portion. Acetyl-CoA may function catalytically in the pyruvate carboxylation reaction by acting as the initial CO2 acceptor, followed by transcarboxylation of malonyl-CoA with pyruvate as shown by Swick and Wood (3). However, the possibility that free malonyl-CoA is an intermediate is doubtful since the carboxylation of acetyl-CoA in the absence of pyruvate is very slow (Table I, Line 2) and direct tests for transcarboxylation reactions with free intermediates have been negative. The role of acetylCoA therefore remains obscure. Malic enzyme is not present in the starting material used in these studies and although phosphoenolpyruvate carboxykinase is present in the crude extracts, it can be separated from the pyruvate carboxylase system. The intracellular location of the pyruvate carboxylase, its relatively high activity, and its apparent high affinity for CO2 suggest that this system coupled with phosphoenolpyruvate carboxykinase may play a part in the synthesis of phosphoenolpyruvate from pyruvate, particularly in mitochondria.