NUCLEOTIDE PYROPHOSPHATASE

Abstract

The wide-spread occurrence of enzymes which split diphosphopyridine nucleotide (DPN) has been appreciated for many years (l), but investigation of the mechanisms of cleavage has been undertaken only recently.In rabbit tissues, two distinct pathways for the breakdown of DPN exist (2).DPN nucleosidase, described by Handler and Klein (3), catalyzes the cleavage of the glycosidic bond between nicotinamide and ribose.DPN pyrophosphatase (2), on the other hand, catalyzes the cleavage of the pyrophosphate bond between adenylic acid and nicotinamide mononucleotide (NMN).Nicotinamide specifically inhibits the nucleosidase and provides a simple way of distinguishing between these two modes of DPN breakdown.An enzyme has been purified from potato extracts that splits the pyrophosphate linkage of DPN and also those of flavin-adenine dinucleotide (FAD), triphosphopyridine nucleotide (TPN), and adenosine triphosphate (ATP) (4).This enzyme has made NMN and riboflavin phosphate readily available for use in studies of the biosynthesis of DPN (5, 6) and FAD (7) and has provided new information concerning the structure of TPN (4).In this report a detailed study of the purification and properties of nucleotide pyrophosphatase is presented. Methods Materials-DPN,reduced DPN (DPNH2), TPN, ATP, adenosine diphosphate (ADP), FAD,' adenosine-5-phosphate, and glycylglycine are described in subsequent papers (6, 7).NMN was prepared as described below.Adenosine-3-phosphate was a Schwarz product, glycerophosphate (01, 52 per cent) an Eastman Kodak product, and thiamine pyrophosphate (cocarboxylase) a Merck sample.GlucoseB-phosphate (8)z and dl-isocitric acid (9)3 were synthetic materials.Crystalline alcohol dehydrogenase was prepared from bakers' yeast according to Racker,4 lactic dehydrogenase was purified from rabbit muscle,