Purification and some properties of inorganic pyrophosphatase from human erythrocytes.

Abstract 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.

• Publication year

  1967

• Venue

  Journal of Biological Chemistry

• Publication date

  1967-05-10

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(18)96026-6PMID 6022858
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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