title: D-myo-inositol 1,4,5-trisphosphate inhibits binding of phospholipase C-delta 1 to bilayer membranes.
abstract: The binding of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) to bilayer membranes composed of phosphatidylcholine (PC) and phosphatidylinositol 4,5-bisphosphate (PIP2) was measured in the presence or absence of inositol phosphates. Binding was inhibited by the natural D-isomer of myo-inositol 1,4,5-trisphosphate (D-InsP3), but not by the L-isomer. The concentration of D-InsP3 required to decrease binding by 50% was 5.4 +/- 0.5 microM. 1-(alpha-Glycerophosphoryl)-D-myo-inositol 4,5-bisphosphate and D-myo-inositol 2,4,5-trisphosphate were nearly as effective as D-Ins(1,4,5)P3. D-myo-inositol monophosphate with phosphate esterified at either positions 1 or 2 of the myo-inositol ring, had no significant effect on binding. D-myo-inositol 1,4-bisphosphate weakly inhibited the binding, whereas the 4,5-isomer was nearly as potent as D-InsP3. Neither ATP nor inorganic phosphate significantly affected binding. As expected, D-Ins(1,4,5)P3 but not L-Ins(1,4,5)P3 decreased the initial rate of PIP2 hydrolysis in bilayer vesicles. The concentration required to decrease hydrolysis by 50% was 12.4 +/- 0.5 microM. A catalytic fragment of PLC-delta 1 that lacks a domain necessary for high affinity PIP2 binding was prepared as previously described (Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593). In contrast to the native enzyme, the rate of PIP2 hydrolysis, catalyzed by the fragment, was not affected by D-Ins(1,4,5)P3. These data suggest that high affinity binding of the enzyme to PIP2 and processive catalysis, involve specific recognition of the 4- and 5-position phosphates of the inositol ring. Our results are consistent with feedback inhibition by the polar head group product, D-Ins(1,4,5)P3, at a step that precedes catalysis, namely interfacial recognition.
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
authors.forename: M E
authors.initials: ME
authors.lastname: Cifuentes
authors.forename: T
authors.initials: T
authors.lastname: Delaney
authors.forename: M J
authors.initials: MJ
authors.lastname: Rebecchi
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D007295:Inositol Phosphates; D007527:Isoenzymes; D008051:Lipid Bilayers; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D015544:Inositol 1,4,5-Trisphosphate; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D043265:Phosphatidylinositol Diacylglycerol-Lyase