The phosphatidyl-myo-inositol-4,5-biphosphate phosphatase from Crithidia fasciculata.
The phosphatase which specifically removes one phosphate group from phosphatidyl-myo-inositol 4,5-bisphosphate was purified up to 6000-fold from the cytosol of the protozoan Crithidia fasciculata. Lipoproteins which interfere with the purification were precipitated by reducing the pH to 4.5. The enzyme was isolated from the supernatant by ammonium sulfate fractionation, gel filtration (Sepharose CL-6B), ion-exchange chromatography (DEAE-Sepharose CL-6B), and hydrophobic chromatography on detergent-saturated phenyl-Sepharose CL-4B. The preparations had specific activities of 44-110 mumol . min-1 . mg protein-1 with phosphatidyl-myo-inositol 4,5-bisphosphate, but were inactive with a variety of lipid and nonlipid phosphate esters. The enzyme was stable in the presence of salt and exhibited a relative mass of 117 000. It formed larger aggregates in the absence of salt and was dissociated into monomers of relative mass 57 000 by sodium dodecyl sulfate. Addition of Triton X-100 to the assay mixture reduced the dependence upon moderation of the charge of the substrate by cetyltrimethylammonium bromide. In the presence of both detergents the Mg2+ dependence of the enzyme was reduced (Km for Mg2+ = 40 microM) while the "apparent" Km for the substrate was unchanged at 240 microM. Substrate precipitation at higher Mg2+ concentrations was eliminated.
D000818:Animals; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003421:Crithidia / Q000201:enzymology*; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D010716:Phosphatidylinositols / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D000099182:Phosphoinositide 5-Phosphatases
D010716:Phosphatidylinositols; D010744:Phosphoric Monoester Hydrolases; D000099182:Phosphoinositide 5-Phosphatases