Specific phospholipid requirements for the solubilization and reconstitution of D-1 dopamine receptors from striatal membranes.
We have reported the solubilization and reconstitution of functional D-1 dopamine receptors from rat striatal tissue, using sodium cholate as detergent [Sidhu, A. (1988) Biochemistry 27, 8768-8776]. Critical to our method of extraction was the absolute requirement for the persistent presence of a crude extract of phospholipids (PLs) from bovine brain, during both solubilization of membranes and reconstitution of the soluble extract into PL vesicles. In the absence of PLs, fewer than 10% of the receptors were recovered, while in the presence of PLs, 40% of the receptors were reconstituted into vesicles. To probe the composition of PLs required by D-1 dopamine receptors during these extraction procedures, specific PLs of a defined composition were used during either solubilization or reconstitution alone or during both solubilization and reconstitution. Phosphatidylcholine (PC), when used during the solubilization procedure alone or during both solubilization and reconstitution, resulted in recovery of 41-48% of the D-1 dopamine receptors but was 3.7-fold less effective when present during reconstitution alone (11%). Phosphatidylethanolamine (PE), when used during reconstitution alone, resulted in recovery of nearly 25% of the D-1 dopamine receptors. When PE was present during either solubilization or both solubilization and reconstitution, 6-11% of the receptors were recovered. If PE was used with PC in ratios of 1:1 or 2:1, respectively, 28-38% of the receptors were recovered. When PL vesicles of PE:PC were present in ratios of 1:2 during both solubilization and reconstitution, the maximum theoretical (74-87%) recovery of total receptor binding sites was achieved.(ABSTRACT TRUNCATED AT 250 WORDS)
Balen
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P
Department of Pediatrics, Georgetown University Children's Medical Center, Georgetown University, Washington, D.C. 20007.
Kimura
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K
Sidhu
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A
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D019826:Cholic Acid; D002793:Cholic Acids; D003342:Corpus Striatum / Q000737:chemistry*; D008081:Liposomes / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology*; D017447:Receptors, Dopamine D1 / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D002793:Cholic Acids; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010743:Phospholipids; D017447:Receptors, Dopamine D1; D019826:Cholic Acid