Purification and some properties of inorganic pyrophosphatase from human erythrocytes.

Inorganic pyrophosphatase was purified BOO-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-j M. Magnesium was essential for activity with an optimal Mgzf PPi ratio of 1. These data suggest that the natural substrate for the erythrocyte pyrophosphatase is MgPPf. 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.