ENZYMATIC CLEAVAGE OF DIPHOSPHOPYRIDINE NUCLEOTIDE WITH RADIOACTIVE PYROPHOSPHATE

Abstract

The reaction of nicotinamide mononucleotide (nicotinamide ribose phosphate) with adenosinetriphosphate (ATP), catalyzed by a specific enzyme in liver and yeast, has been shown to result in the production of diphosphopyridine nucleotide (DPN) and inorganic pyrophosphate (PP) (1).Similarly, the reaction of riboflavin phosphate with ATP, catalyzed by another yeast enzyme, produces flavin-adenine dinucleotide and PP (2).These reactions are freely reversible; cleavage of the dinucleotides by PP results in the formation of ATP and the respective mononucleotides.No information is available, however, as to the exact way in which the phosphate is distributed in these molecular rearrangements.A hypothetical mechanism is one resembling the reversible splitting of polysaccharides, disaccharides, and nucleosides by inorganic orthophosphate.Evidence to support such a "pyrophosphorylase" action has now been obtained by studies with radioactive PP.The reaction may be formulated as (1) PaPa2 + nicotinamide-ribose-P-P-adenosine (DPN) d nicotinamide-ribose-P + P32P3s-P-adenosine (ATP) MethodsMateriaZs-P32P32 was prepared by dehydration of NazHP3204.H3P3204 in acid solution was obtained from the Oak Ridge National Laboratory and had a stated specific activity of 0.025 mg. per mc.and a stated radiochemical purity of 99.9 per cent.0.2 ml.(0.77 mc.) was adjusted with NaOH to about pH 9, added to 0.40 ml. of 0.1 M Na2HP04 in a platinum crucible, dried at llO", and then heated at 225" for about 24 hours to constant weight.The product contained 0.02 mole of orthophosphate per mole of phosphate; 99 per cent was hydrolyzable by 1 N HCl in 10 minutes at 100" and by purified yeast inorganic pyrophosphatase.Chromatographic analysis on Dowex-1 (chloride form) with 0.05 M NaCl in 0.01 N HCl as the eluant indicated that 1.7 per cent of the counts were in the orthophosphate region of the elution curve and 86.8 per cent in the PP region.Some preparations, perhaps as a result of overheating or incorrect pH adjustment, contained polyphosphate, determined as acid-labile phos-