Nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei. The effects of nicotinamide mononucleotide concentration and pH on dinucleotide synthesis

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Abstract
Nicotinamide mononucleotide was prepared from nicotinamide-adenine nucleotide by hydrolysis with nucleotide pyrophosphatase or by pyrophosphorolysis with nicotinamide mononucleotide adenylyltransferase from pig-liver nuclei. The product was characterized by polarimetry, spectrophotometry and potentiometric titration. The rate of formation of nicotinamide-adenine dinucleotide from adenosine triphosphate (4 m[image]) and nicotinamide mononucleotide (at 5 or 6 concentrations from 0.14 to 1.4 m[image]) in the presence of the adenylyltransferase and of magnesium chloride (15 m[image]) was measured at 25[degree] and at 17 pH values from 4.97 to 10.55. With the assay system used, the rate of formation of nicotinamide-adenine dinucleotide did not vary during the period of incubation by more than 1%. A statistical method was used to evaluate the Michaelis constants (Km) and the apparent maximum rates (V[image], at infinite nicotinamide mononucleotide concentration) for each pH. A further statistical analysis of the variation of Km with pH indicated the presence of dissociating groups, with pK values 5.76 [plus or minus] 0.048 and 9.97 [plus or minus] 0.069, which participated in the interaction of nicotinamide mononucleotide with the enzyme-adenosine triphosphate-magnesium complex. For the effective ionic species, p[image]m was 4.10 + 0.019. From a consideration of the variation of pKm with pH and of log V[image] with pH, possible mechanisms of the synthesis of nicotin-amide-adenine dinucleotide were formulated.