Purification and Some Properties of Inorganic Pyrophosphatase from Human Erythrocytes

Inorganic pyrophosphatase was purified 1800-fold from human erythrocytes by a procedure involving complete hemolysis of the cells, removal of hemoglobin, ammonium sulfate fractionation, diethylaminoethyl cellulose column chromatography, and gel filtration. Magnesium chloride and 2-mercaptoethanol stabilized the activity. The procedure yielded an enzyme with an optimal pH of 7.7, and an apparent Michaelis constant of 9.7 x 10-6 m. Magnesium was essential for activity with an optimal Mg2+ PPi ratio of 1. These data suggest that the natural substrate for the erythrocyte pyrophosphatase is MgPP2-i. The enzyme showed very strict specificity for inogranic pyrophosphate. Adenosine triphosphate, adenosine diphosphate, inosine triphosphate, and inorganic tripolyphosphate were not acted upon in the presence of Mg2+ or Zn2+ as activator. Moreover, unlike the mitochondrial and the microsomal pyrophosphatases, the erythrocyte enzyme showed no PPi-glucose transphosphorylase activity. The Arrhenius plot of the erythrocyte pyrophosphatase exhibited a transition at 29° with an activation energy of 8,560 cal per mole above this temperature and 12,900 cal per mole below it. The activity was inhibited by p-hydroxymercuribenzoate, but was much less sensitive to alloxan, N-ethylmaleimide, and iodoacetamide. In the erythrocyte, inorganic pyrophosphatase probably plays a role in nicotinamide adenine dinucleotide formation by hydrolyzing the PPi produced in its three synthetic steps.