Assay of phosphoenolpyruvate carboxykinase in crude yeast extracts.

The applicability of a spectrophotometric assay of phosphoenolpyruvate car?ykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate → oxalacetate + ATP; oxalacetate + NADH → malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn²⁺. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 mm quinolinate, which is a known inhibitor of liver phosphoenolpyruvate car?ykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate car?ykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced baker's yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.