NEUROSPORA DIPHOSPHOPYRIDINE NUCLEOTIDASE

Abstract

The cleavage by specific enzymes of diphosphopyridine nucleotide (DPN) at a number of different points in the coenzyme molecule is now well established.A splitting of DPN at the nicotinamide-ribose linkage in animal tissues has been described by Handler and Klein (1).This enzyme, which is usually referred to as DPN nucleosidase, has recently been studied in more detail in brain by McIllwain and Rodnight (2).Kornberg and Pricer have studied and purified an enzyme from potatoes which hydrolyzes DPN into adenylic acid and nicotinamide mononucleotide (3).A similar enzyme apparently also occurs in animal tissues (4).Kornberg (5) has also found an enzyme in yeast and liver which induces the reversible pyrophosphorolysis of DPN into adenosinetriphosphate and nicotinamide mononucleotide.An enLyme from Neurospora crassa has been detected which attacks the nicotinamide-ribose linkage of DPN (6).This enzyme appears in high concentrations in Neurospora grown on a zinc-deficient medium.The present paper deals with the properties and purification of this enzyme, and the products which result from the enzymatic action. Methods Materials-TheDPN used in these studies was obtained from the Schwarz Laboratories.The purity was 53 per cent, as determined by absorption at 340 rnp after complete enzymatic reduction (7).The triphosphopyridine nucleotide (TPN) used in these studies was a preparation of 50 per cent purity.The nicotinamide mononucleotide was prepared by the action of potato nucleotide pyrophosphatase on DPN.Nicotinamide riboside was prepared by the action of intestinal phosphatase on DPN.Reduced DPN was prepared by mixing 5 ml. of a 0.4 per cent solution of DPN with 1 ml. of 0.5 M NaHC03 and 0.75 ml. of freshly prepared 1 per cent sodium hydrosulfite in a Thunberg tube.After incubation in vcxuo at 37" for 1 hour, the tube was chilled in ice before opening.0.2 ml.