The participation of inorganic pyrophosphate in the reversible enzymatic synthesis of diphosphopyridine nucleotide.

* For DPN synthesis, 1.0 ml. of reaction mixture contained 50 y of the enzyme preparation, 0.3 micromole of MgCl 2, and 50 micromoles of glycylglycine buffer (pH 7.4) in addition to ATP and NMN; for ATP synthesis 1.0 ml. contained 50 y of the enzyme preparation, 0.75 micromole,of MgC12, and 50 micromoles of glycylglycine buffer (pH 7.4) in addition to DPN and P-P. Constant values were reached after 30 to 40 minutes at 38”. t ATP was estimated spectrophotometrically by triphosphopyridine nucleotide reduction in the presence of glucose, hexokinase, and Zwischenferment, and DPN by reduction with the triose phosphate dehydrogenase system. Inorganic pyrophosphate was estimated as orthophosphate after acid hydrolysis of the precipitated and washed manganous salt and acid-labile phosphate as the orthophosphate released after 10 minute hydrolysis in 1 NH~SO~ at 100”. 1 NMN was prepared by hydrolysis of DPN with nucleotide pyrophosphatase.’ 2 After purification the ratio, nicotinamide-ribose moiety to organic phosphate, was 1.0.