title: Evidence for a regulatory role of cholesterol superlattices in the hydrolytic activity of secretory phospholipase A2 in lipid membranes.
abstract: We have conducted a detailed study of the effect of membrane cholesterol content on the initial hydrolytic activity of Crotalus durissus terrificus venom phospholipase A2 (sPLA2) in large unilamellar vesicles of cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and cholesterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC) at 37 degrees C. The activity was monitored by using the acrylodan-labeled intestinal fatty acid binding protein and HPLC. In contrast to conventional approaches, we have used small cholesterol concentration increments ( approximately 0.3-1.0 mol %) over a wide concentration range (e.g., 13-54 mol % cholesterol). In both membrane systems examined, the initial hydrolytic activity of sPLA2 is found to change with cholesterol content in an alternating manner. The activity reaches a local minimum when the membrane cholesterol content is at or near the critical cholesterol mole fractions (e.g., 14.3, 15.4, 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol) predicted for cholesterol regularly distributed in either hexagonal or centered rectangular superlattices. According to the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073; Liu et al. (1997) Biophys. J. 72, 2243-2254], the extent of lipid superlattices is maximal at the critical cholesterol mole fractions, at which the membrane free volume is minimal. Thus, our present data can be taken to indicate that the initial hydrolytic activity of sPLA2 is governed by the extent of cholesterol superlattice. These data provide the first functional evidence for the formation of cholesterol superlattices in both saturated (e.g., DMPC) and unsaturated (e.g., POPC) liquid-crystalline phospholipid bilayers. The data also illustrate the functional importance of cholesterol superlattice and demonstrate a new type of regulation of sPLA2. Furthermore, upon binding to cholesterol/POPC large unilamellar vesicles, the intrinsic fluorescence intensity of sPLA2 shows an alternating variation with cholesterol content, exhibiting a minimum at the critical cholesterol mole fractions. This result suggests that either the number of sPLA2 bound to lipid vesicles or the conformation of membrane-bound sPLA2 or both vary with the extent of the cholesterol superlattice in the plane of the membrane.
authors: Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors: F
authors: F
authors: Liu
authors: P L
authors: PL
authors: Chong
mesh_terms: D001665:Binding Sites; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004789:Enzyme Activation; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
chemical_list: D003435:Crotalid Venoms; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D002784:Cholesterol; D010741:Phospholipases A; D054467:Phospholipases A2; C017251:1,2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine