573 Effect of ethanol on the di- and triphosphopyridine nucleotide content in rat liver

Abstract

This chapter discusses the determination of pyridine nucleotide transhydrogenase. The usual assay for the enzyme involves the reduction of DPN by TPNH (reaction 1). The TPN is usually present in catalytic amounts and TPNH is generated by the TPN-specific isocitric dehydrogenase from pig heart. The reduced TPN in the presence of the transhydrogenase then reduces the DPN. Some transhydrogenases, such as the enzyme from hog brain, catalyzes an exchange between the oxidized and reduced forms of DPN. This type of transfer can be followed by the use of deamino-DPN as an oxidizing agent for reduced DPN (reaction 2). Assay methods for both reactions are described. A unit of activity is defined as the amount of transhydrogenase which will produce an optical-density change of 0.01 in 1 to 4 minutes after addition of the acceptor nucleotide (DPN in reaction 1, deamino-DPN in reaction 2). The assay procedures can be used in both crude tissue and bacterial extracts. However, the addition of 3 micromoles of KCN should be included to prevent reoxidation of the reduced nucleotides. In the case of the animal preparations, it is essential to add 60 micromoles of nicotinamide to prevent splitting of the oxidized nucleotides by DPNase. The chapter also discusses the purification procedure for Pseudomonas transhydrogenase and pyridine nucleotide transhydrogenase from beef heart.