ENRICHMENT OF SATURATED FATTY-ACID CONTAINING PHOSPHOLIPIDS IN SHEEP BRAIN-SEROTONIN RECEPTOR PREPARATIONS - USE OF MICROWAVE IRRADIATION FOR RAPID TRANSESTERIFICATION OF PHOSPHOLIPIDS

During enrichment of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-binding serotonin 5-HT1A receptors from sheep brain gray matter (membrane isolation, detergent solubilization and reconstitution into vesicles) a consistent and striking increase in the composition of saturated fatty acids was observed in phospholipids which were coisolated with the receptors. A rapid procedure has been developed for the methylation of free and phospholipid linked fatty acids which were thus analyzed by gas chromatography-mass spectrometry (GC/MS). Esterification of free fatty acids and transesterification of phospholipid linked fatty acids were achieved with 14% boron trifluoride in methanol (BF3-CH3OH) in 20 s and 50 s, respectively, under low power microwave irradiation (60 W) with a post-reaction cooling of < 5 min. This is in contrast to the conventional method of heating in a boiling water bath for 10-15 min with BF3-CH3OH which is inevitably preceded by time-consuming and inconvenient clamping of vials and followed by cooling for 10 min before the vials can be safely opened. Analysis of fatty acid profiles in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) from egg yolk, phosphatidylinositol (PI) from bovine liver and phosphatidylserine (PS) from bovine brain by both techniques showed comparable results. During detergent solubilization of sheep brain gray matter, the overall proportion of saturated fatty acids in PE (major lipid), PI, PC (major lipid) and PS increased from 50-60% in sheep brain phospholipids to 70-75% in 1.5% CHAPS solubilized, reconstituted and biologically active serotonin 5-HT1A preparations. In sharp contrast, the proportions of saturated fatty acids in 1.5% Triton X-100 solubilized PE (48.1%) (major lipid), PI (63.6%), PC (60.6%) (major lipid) and PS (62.2%) were not significantly different from those in the original sheep brain membranes. Strikingly, this was coupled with the occurrence of very low levels of 5-HT1A receptor activity in the Triton X-100 solubilized preparations. The abundance of 5-HT1A sites in the enriched vesicles obtained only from the CHAPS-solubilized preparations was further confirmed by specific radiolabeling of a 58-kDa polypeptide by the 5-HT1A specific ligand p-aminophenylethyl-m-trifluoromethylphenylpiparazine (PAPP) which was coupled to a I-125-labeled, photoreactive, heterobifunctional cross-linker, sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Thus CHAPS-solubilized 5-HT1A receptor preparations are depleted in the more rigid lipids such as sphingolipids and cholesterol, (Banerjee et al. (1990) Biochim. Biophys. Acta 1044, 305-314), but are enriched in vesicle-stabilizing, phospholipid-linked saturated fatty acids which in turn probably stabilize the heptahelical, membrane bound 5-HT1A receptor.