Differential solubilization of membrane lipids by detergents: coenrichment of the sheep brain serotonin 5-HT1A receptor with phospholipids containing predominantly saturated fatty acids.
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.
Banerjee
P
P
Department of Pediatrics, University of Chicago, Illinois 60637.
Dasgupta
A
A
Chromy
B A
BA
Dawson
G
G
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
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