INHIBITION OF PHOSPHOLIPASE C-DELTA(1) CATALYTIC ACTIVITY BY SPHINGOMYELIN

We measured the ability of sphingomyelin (SPM) to inhibit phosphatidyl inositol 4,5-bisphosphate [PI(4,5)P-2] hydrolysis catalyzed by human p hospholipase C-delta(1) (PLC-delta(1) in model membranes and detergent phospholipid mixed micelles. SPM strongly inhibited PLC-delta(1) cata lytic activity measured in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine IPC), PI(4.5)P-2, and SPM from brain or egg . At 37 or 45 degrees C, the rate of PI(4,5)P-2 hydrolysis in PC/SPM/P I(,5)P-2 vesicles (15:80:5 mol:mol) was less than 25% of that observed in PC/PI(4,5)P-2 vesicles (95:5), By contrast, catalysis was only wea kly inhibited by equivalent concentrations of the SPM analog, 3-deoxy- 2-O-stearoyl-SPM, which lacks hydrogen bond-donating groups at the C-3 and C-2 positions of the sphingolipid backbone. Inhibition by SPM was not observed in detergent/phospholipid mixed micelles, The binding af finity of PLC-delta(1) for vesicles containing PC and PI(4,5)P-2 was s lightly diminished by inclusion of SPM in the lipid mixture, but not e nough to account for the decreased rate of catalysis. We could find no evidence of specific binding of the enzyme to SPM, which argues again st a simple negative allosteric mechanism. To understand the cause of inhibition, the effects of SPM and 3-deoxy-2-O-stearoyl-SPM on the bul k properties of the substrate bilayers were examined. Increasing the m ole fraction of SPM altered the fluorescence emission spectra of two s ets of head group probes, 6-lauronyl(N,N-dimethylamino)naphthalene and 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, that are sensitive to water content at the membrane/solution interface, Results obtained with both probes suggested a reduction in hydration with increasing S PM content, Vesicles containing 3-deoxy-2-O-stearoyl-SPM produced inte rmediate changes. Our results are most consistent with a model in whic h SPM inhibits PLC by increasing interlipid hydrogen bonding and by de creasing membrane hydration; both factors raise the energy barrier for activation of PLC-delta(1) at the membrane/protein microinterface.