title: Differential solubilization of membrane lipids by detergents: coenrichment of the sheep brain serotonin 5-HT1A receptor with phospholipids containing predominantly saturated fatty acids.
abstract: Most membrane receptors lose binding activity during purification and we studied the correlation between this event and differential solubilization of membrane lipids by detergents. Both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [Chaps; high critical micellar concentration (cmc) approximately 0.5%] and n-dodecyl beta-D-maltoside (DDM; low cmc approximately 0.009%) solubilized the 8-[3H]hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT)-binding serotonin 5-HT1A receptor (5-HT1A-R) optimally at 2% (w/v) detergent concentration despite the widely differing cmc of the two detergents. In contrast, n-octyl-beta-D-glucopyranoside (octyl glucoside; high cmc approximately 0.7%), Thesit (low cmc approximately 0.005%), and Triton X-100 (low cmc approximately 0.013%) solubilized virtually no [3H]8-OH-DPAT-binding activity. The total mass of solubilized lipids was always low at 0.5% detergent concentration and attained a plateau between 1 and 2.5% for all detergents except octyl glucoside. The mass of octyl glucoside-solubilized lipids showed an increasing trend even at 3.0% detergent concentration. The total amount of solubilized lipid is unrelated to the amount of 5-HT1A-R solubilized but the species of lipid is important. Thus Chaps and DDM, with diverse structures and cmc, both preferentially solubilized phospholipids enriched in saturated fatty acids (67 and 72%, respectively). In contrast, octyl glucoside, Triton X-100, and Thesit showed no preference in solubilizing phospholipids. Octyl glucoside, which solubilized significantly higher proportions of saturated fatty-acid-containing phosphatidylethanolamine (slightly higher saturated fatty acids in total phospholipids), also produced more (twofold higher) solubilized 5-HT1A sites than Triton X-100 and Thesit. This suggests an optimum involvement of saturated fatty acid side chains in forming tightly packed vesicles which stabilize the 5-HT1A-R more than the vesicles of higher fluidity formed by phospholipids containing higher proportions of cis-double-bonded unsaturated fatty acids. Indeed, delipidation of the 1.5% Chaps-solubilized receptor preparations by Sephacryl S-200 column chromatography eliminated essentially all [3H]8-OH-DPAT-binding activity. Therefore, for efficient recovery during solubilization and reconstitution of a prototypic heptahelical receptor (5-HT1A), it is essential to stabilize the receptor protein through association with saturated phospholipids.
authors.affiliation: Department of Pediatrics, University of Chicago, Illinois 60637.
authors.forename: P
authors.initials: P
authors.lastname: Banerjee
authors.forename: A
authors.initials: A
authors.lastname: Dasgupta
authors.forename: B A
authors.initials: BA
authors.lastname: Chromy
authors.forename: G
authors.initials: G
authors.lastname: Dawson
mesh_terms: D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000818:Animals; D001923:Brain Chemistry; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D005227:Fatty Acids / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D005960:Glucosides; D008563:Membrane Lipids / Q000032:analysis* / Q000737:chemistry; D010743:Phospholipids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011985:Receptors, Serotonin / Q000032:analysis* / Q000378:metabolism; D012756:Sheep; D012995:Solubility
chemical_list: D002793:Cholic Acids; D003902:Detergents; D005227:Fatty Acids; D005960:Glucosides; D008563:Membrane Lipids; D010743:Phospholipids; D011985:Receptors, Serotonin; C040358:dodecyl maltoside; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate