Human erythrocyte cytosol phosphatidyl-inositol-bisphosphate phosphatase

Abstract

A phosphatidyl- myo-inositol-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 (NH 4) 2SO 4 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-P 2 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. Mg 2+ was required (‘apparent’ K m = 12 μM). The ‘apparent’ K m for the substrate was 0.27 mM and the specific activity was 765 ± 191 (S.D.) nmol/min per mg protein. Inhibition by Ca 2+ (‘apparent’ K i = 50 μM) was competitive with Mg 2+. 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 than 1% (w/v) and by agents which react with sulfhydryl groups, but was unaffected by dithioerythritol and F −.