Human erythrocyte cytosol phosphatidyl-inositol-bisphosphate phosphatase

Biochim Biophys Acta. 1981 Oct 13;661(2):323-33. doi: 10.1016/0005-2744(81)90021-8.

Abstract

A phosphatidyl-myoinositol-4,5-bisphosphate phosphohydrolase (phosphatidyl-inositol-bisphosphate phosphatase, EC 3.1.3.36) was detected in human erythrocytes and partially purified from the cytosol. Hemoglobin was removed by (NH4)2SO4 fractionation and chromatography on CM-Sepharose CL-6B. A 27,000-fold purification was achieved following gel filtration,, ion-exchange chromatography and hydrophobic chromatography. Although the preparation was not homogeneous, the molecular mass of the enzyme was estimated to be 105,000 by gel filtration. The activity was stabilized by a non-ionic detergent (Triton X-100). The enzyme was active with PI-P2 and, to a lesser extent, myo-inositol 1, 4, 5-trisphosphate but not with PI-P nor a variety of other lipid and non-lipid phosphate esters. In the presence of both cationic and non-ionic detergents, the effects of divalent cations were independent of substrate concentration. Mg2+ was required ('apparent' Km = 12 muM). The 'apparent' Km for the substrate was 0.27 mM and the specific activity was 765 +/- 191 (S.D.) nmol/min per mg protein. Inhibition by Ca2+ ('apparent' Ki = 50 microM) was competitive with Mg2+. Neomycin was an inhibitor at 10(-6) - 10(-4) M but only in the absence of Triton X-100. The phosphatase was inhibited by hemoglobin at concentrations higher that 1% (w/v) and by agents which react with sulfhydryl groups, but was unaffected by dithioerythritol and F-.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cetrimonium
  • Cetrimonium Compounds / pharmacology
  • Cytosol / enzymology
  • Erythrocytes / enzymology*
  • Fractional Precipitation
  • Humans
  • Hydrogen-Ion Concentration
  • Magnesium / pharmacology
  • Molecular Weight
  • Octoxynol
  • Phosphatidylinositols / antagonists & inhibitors
  • Phosphatidylinositols / blood
  • Phosphatidylinositols / isolation & purification
  • Phosphoric Monoester Hydrolases / antagonists & inhibitors
  • Phosphoric Monoester Hydrolases / blood*
  • Phosphoric Monoester Hydrolases / isolation & purification
  • Polyethylene Glycols / pharmacology
  • Substrate Specificity

Substances

  • Cetrimonium Compounds
  • Phosphatidylinositols
  • Polyethylene Glycols
  • Octoxynol
  • Phosphoric Monoester Hydrolases
  • phosphoinositide 5-phosphatase
  • Magnesium
  • Cetrimonium