Improvement in conditions for solubilisation and characterisation of brain D2 dopamine receptors using various detergents.
A series of detergents of varying chemical properties has been tested for solubilisation of bovine caudate nucleus D2 dopamine receptors using [3H]spiperone binding to assay the solubilised sites. The properties of the lysophosphatidylcholine (LPC)- and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulphonate (CHAPS)-solubilised preparations are described in detail. The preparations are truly solubilised, and sucrose density gradient and gel filtration data are reported. Specific [3H]spiperone binding in the LPC-solubilised preparation assayed at 4 degrees C is solely to D2 dopamine receptors. If the assay temperature is raised to 25 degrees C, the amount of specific [3H]spiperone binding is largely unchanged, but it forms a greater proportion of the total [3H]spiperone binding owing to a reduction in nonstereospecific (spirodecanone) [3H]spiperone binding at the higher temperature. The effect of raising the assay temperature is important as it enables more precise determinations of specific [3H]spiperone binding to be made. Part of the specific [3H]spiperone binding at 25 degrees C is to solubilised S2 serotonin receptors in addition to D2 dopamine receptors. Good correlations are observed between the affinities for binding of ligands to the solubilised D2 receptors and corresponding data obtained on membrane-bound receptors. Agonist binding in LPC-solubilised preparations is insensitive to guanine nucleotides. It is speculated that the spirodecanone sites represent, in part, proteolysed or damaged D2 dopamine, or S2 serotonin, receptors. In the CHAPS-solubilised preparation the pharmacological profile of [3H]spiperone binding is unclear when assayed at 4 degrees C, but in assays at 25 degrees C a clear serotonin S2 receptor component of specific [3H]spiperone binding can be discerned.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002417:Cattle; D002421:Caudate Nucleus / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D019826:Cholic Acid; D002793:Cholic Acids; D003902:Detergents; D004072:Digitonin; D006150:Guanine Nucleotides / Q000494:pharmacology; D008244:Lysophosphatidylcholines; D011480:Protease Inhibitors / Q000494:pharmacology; D011954:Receptors, Dopamine / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D012965:Sodium Chloride; D012995:Solubility; D013134:Spiperone / Q000378:metabolism; D013501:Surface-Active Agents
D002793:Cholic Acids; D003902:Detergents; D006150:Guanine Nucleotides; D008244:Lysophosphatidylcholines; D011480:Protease Inhibitors; D011954:Receptors, Dopamine; D013501:Surface-Active Agents; D012965:Sodium Chloride; D013134:Spiperone; D019826:Cholic Acid; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate