Purification and Properties of Reduced Diphosphopyridine Nucleotide Kinase from Yeast Mitochondria

Abstract

Abstract An enzyme that phosphorylates DPNH to form TPNH has been isolated from aerobic yeast mitochondria and purified 127-fold. This enzyme differs from other reported DPN kinases in that it is specific for DPNH as a substrate and requires high concentrations of a carboxylic acid such as acetate for maximal activity. In the presence of 0.2 m sodium acetate, the apparent Km values for DPNH, ATP, and Mg++ are 42 µm, 1.0 mm, and 1.0 mm, respectively. Purified DPNH kinase is highly unstable; however, it can be protected from denaturation by 0.2 m ammonium sulfate. Ammonium sulfate itself does not activate the enzyme. Mitochondrial DPNH kinase represents about 5% of the total DPN and DPNH kinase activities of the yeast cell and appears to be involved in maintaining the level of triphosphopyridine nucleotide in the mitochondrion.