Human erythrocyte cytosol phosphatidyl-inositol-bisphosphate phosphatase.
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-.
D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004912:Erythrocytes / Q000201:enzymology*; D005590:Fractional Precipitation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D017830:Octoxynol; D010716:Phosphatidylinositols / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity; D000099182:Phosphoinositide 5-Phosphatases
D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D008274:Magnesium; D017830:Octoxynol; D010716:Phosphatidylinositols; D010744:Phosphoric Monoester Hydrolases; D011092:Polyethylene Glycols; D000099182:Phosphoinositide 5-Phosphatases