The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.Keywords: Bacillus cereus; phospholipase C ; gangliosides ; interfacial catalysis.Phospholipase C (PLC) from Bacillus cereus has been shown to catalytically induce unilamellar vesicle aggregation, lipid bilayer merging and fusion [l, 21. The molecular mechanism appears to involve isotropic lipid structures during the fusion process 131. It is also known that complex glycosphingolipids such as gangliosides have inhibitory effects on PLC and phospholipase AZ [4-71. In addition, it has been reported that gangliosides markedly inhibit bilayer-bilayer interaction and fusion promoted by basic proteins [S] or by PLC phosphohydrolase activity [2]. These effects are probably mediated by the modulatory properties of low proportions of gangliosides in the formation of non-bilayer phases [7].It is therefore of interest to investigate the effects of gangliosides on the enzymatic activity of B. cereus PLC with the aim of explaining previous findings by which complex gangliosides arrest the fusion triggered by this enzyme [2]. In this work we have studied the action of glycosphingolipids of different complexity on the activity of B. cereus PLC against mixed monolayers, micelles and small unilamellar vesicles.
MATERIALS AND METHODSGlycosphingolipids were purified from bovine brain as described previously [4]. Egg PtdCho, Lau,GroPCho and Pam2- Abbreviations. PLC, phospholipase C ; Dec,GroPCho, didecanoylglycerophosphocholine; Lau,GroPCho, dilauroylglycerophosphocholine; Pam2GroPCho, dipalmitoylglycerophosphocholine; PyDec,GroPcho, 1,2-bis( 1 -pyrenedecanoyl)-sn-glycero-3-phosphocholine ; Cer, N-acylsphingoid; G,,, Gal~1-3Gal-NAc~l-4Ga1(3~2uNeuAc)~1-+4Glc~l -+Cer ; G,,,,, NeuAca2-3Gal~l-3Gal-NAc/J'l-+4Ga1(3~2aNeu-Ac)~1-4Glc~l-Cer; G., I h , NeuAcu2-+3Gal~l~3Gal-Nac~1-4Gal-(3+2aNeuAc8+-2aNeuAC)/J'1-4Glc/J'l+Cer; G,,,,, Galpl+3GalNAc-~l+4Ga1(3~2nNeuAc8+2aNeuAc)~1-4Glc~l+Cer; G,,,, mixture of total brain gangliosides (GM, 21 8; G,,,, 42%; G,,,, 18%; Gr,, 19%); GSLs, glycosphingolipids; Sulfatide, Gal(3+sulfate)/ll-l Cer.Enzymes. Phospholipase C (EC 3.1.4.3); phospholipase A? (EC 3.1.1.4); lipase (EC 3.1.1.3).GroPCho were from Avanti Polar Lipids (Birmingham AL). PyDec,GroPCho and 1 -pyrene-decanoic acid were from Molecular Probes (Eugene...
