Association of alpha-phosphatidylinositol-specific phospholipase C with phospholipid vesicles.
The alpha isoform of phosphatidylinositol-specific phospholipase C (alpha-PI-PLC, Mr 62,000) was purified from bovine brain. Enzyme activity was dependent on calcium, sodium cholate and showed the anticipated specificity for the phosphatidylinositols. Calcium interaction with this protein, investigated by gel filtration chromatography, showed no detectable binding at calcium concentrations adequate to activate the enzyme. Association of alpha-PI-PLC with phospholipid vesicles was studied by light scattering, fluorescence energy transfer and gel-filtration chromatography. The enzyme readily associated with vesicles of high charge density, with vesicles of crude acidic phospholipids and with PIP2. Interaction was characterized by a rapid association followed by slower addition of more protein to the phospholipid. Complexes containing 20-30 percent protein (by weight) were readily obtained. Calcium had only a small effect on this interaction. The protein-phospholipid complexes appeared to bind less calcium than a similar amount of phospholipid alone. Thus, alpha-PI-PLC did not appear to be a calcium-binding protein in either its free or membrane-associated states. Although alpha-PI-PLC showed the highest propensity to bind to phospholipids, a number of other proteins also associated with phospholipids under the conditions used. Thus, whether or not the observed interaction of alpha-PI-PLC with membranes was specific and biologically important or whether it was a process common to many proteins, was not known. Knowledge of this interaction may enhance our understanding of possible mechanisms for protein-membrane interactions in general.
Xu
C J
CJ
Department of Biochemistry, University of Minnesota, St. Paul.
Nelsestuen
G L
GL
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D008564:Membrane Potentials; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010743:Phospholipids / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010743:Phospholipids; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium