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Inhibition of phospholipase C-delta 1 catalytic activity by sphingomyelin. 1996

S Scarlata, and R Gupta, and P Garcia, and H Keach, and S Shah, and C R Kasireddy, and R Bittman, and M J Rebecchi
Department of Anesthesiology, State University of New York at Stony Brook 11794, USA. scarlata@physiology.pnb.sunysb.edu

We measured the ability of sphingomyelin (SPM) to inhibit phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] hydrolysis catalyzed by human phospholipase C-delta 1 (PLC-delta 1) in model membranes and detergent phospholipid mixed micelles. SPM strongly inhibited PLC-delta 1 catalytic activity measured in large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine (PC), PI(4,5)P2, and SPM from brain or egg. At 37 or 45 degrees C, the rate of PI(4,5)P2 hydrolysis in PC/SPM/PI(4,5)P2 vesicles (15:80:5 mol:mol) was less than 25% of that observed in PC/PI(4,5)P2 vesicles (95:5). By contrast, catalysis was only weakly 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 affinity of PLC-delta 1 for vesicles containing PC and PI(4,5)P2 was slightly diminished by inclusion of SPM in the lipid mixture, but not enough to account for the decreased rate of catalysis. We could find no evidence of specific binding of the enzyme to SPM, which argues against a simple negative allosteric mechanism. To understand the cause of inhibition, the effects of SPM and 3-deoxy-2-O-stearoyl-SPM on the bulk properties of the substrate bilayers were examined. Increasing the mole fraction of SPM altered the fluorescence emission spectra of two sets of head group probes, 6-lauronyl(N,N-dimethylamino)naphthalene and N-[5-(dimethylamino)naphthalene-1-sulfonyl]-1,2-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 SPM content. Vesicles containing 3-deoxy-2-O-stearoyl-SPM produced intermediate changes. Our results are most consistent with a model in which SPM inhibits PLC by increasing interlipid hydrogen bonding and by decreasing membrane hydration; both factors raise the energy barrier for activation of PLC-delta 1 at the membrane/protein microinterface.

UI MeSH Term Description Entries
D007527 Isoenzymes Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics. Alloenzyme,Allozyme,Isoenzyme,Isozyme,Isozymes,Alloenzymes,Allozymes
D008051 Lipid Bilayers Layers of lipid molecules which are two molecules thick. Bilayer systems are frequently studied as models of biological membranes. Bilayers, Lipid,Bilayer, Lipid,Lipid Bilayer
D008823 Micelles Particles consisting of aggregates of molecules held loosely together by secondary bonds. The surface of micelles are usually comprised of amphiphatic compounds that are oriented in a way that minimizes the energy of interaction between the micelle and its environment. Liquids that contain large numbers of suspended micelles are referred to as EMULSIONS. Micelle
D010738 Type C Phospholipases A subclass of phospholipases that hydrolyze the phosphoester bond found in the third position of GLYCEROPHOSPHOLIPIDS. Although the singular term phospholipase C specifically refers to an enzyme that catalyzes the hydrolysis of PHOSPHATIDYLCHOLINE (EC 3.1.4.3), it is commonly used in the literature to refer to broad variety of enzymes that specifically catalyze the hydrolysis of PHOSPHATIDYLINOSITOLS. Lecithinase C,Phospholipase C,Phospholipases, Type C,Phospholipases C
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D002417 Cattle Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor. Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006860 Hydrogen Bonding A low-energy attractive force between hydrogen and another element. It plays a major role in determining the properties of water, proteins, and other compounds. Hydrogen Bonds,Bond, Hydrogen,Hydrogen Bond
D006868 Hydrolysis The process of cleaving a chemical compound by the addition of a molecule of water.
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia

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