Large-scale purification of alpha 2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes.
A simple large-scale purification of alpha 2-adrenergic receptor-enriched membranes from human platelets is described. Binding of the antagonist [3H]yohimbine is enriched 3-5-fold compared to a crude membrane fraction. Binding of low concentrations of the partial agonist 3-H-rho-aminoclonidine is increased 15-20-fold due to a higher binding affinity for the purified membranes. A soluble inhibitor of 3H-rho-aminoclonidine binding to purified membranes is found even in thrice-washed crude platelet membranes. The guanine nucleotides GDP and GTP are found to account for this inhibitory activity. Forskolin-stimulated adenylate cyclase activity is also enriched in the purified membrane fraction. Adenylate cyclase activity is inhibited by alpha 2-agonist to a comparable extent in all membrane fractions. This membrane preparation should prove useful in studies of alpha 2-adrenergic receptor mechanisms.
D000262:Adenylyl Cyclases / Q000378:metabolism; D001792:Blood Platelets / Q000648:ultrastructure*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D002499:Centrifugation, Density Gradient; D003000:Clonidine / Q000031:analogs & derivatives / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004837:Epinephrine / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D009638:Norepinephrine / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D014316:Tritium; D015016:Yohimbine / Q000378:metabolism
D011943:Receptors, Adrenergic, beta; D014316:Tritium; D006153:Guanosine Diphosphate; D005576:Colforsin; D015016:Yohimbine; C016986:apraclonidine; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D003000:Clonidine; D009638:Norepinephrine; D004837:Epinephrine