Characterization of 3H-labelled platelet activating factor receptor complex solubilized from rabbit platelet membranes.
Rabbit platelet membranes, preincubated with 3H-labeled platelet activating factor ([3H]PAF), were solubilized with 2% digitonin. Sedimentation of the detergent extract in a sucrose density gradient revealed a major labeled component with a sedimentation coefficient (s20,omega) of 10.5 S, which was substantially diminished when an excess of unlabeled PAF or L-652,731, (trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran), (PAF antagonist) was present in the preincubation mixture, suggesting that the 10.5 S component is a specific receptor-bound [3H]PAF complex. Gel filtration of the [3H]PAF-receptor complex on Sephacryl S-300 revealed a single radiolabeled fraction with an apparent Stokes' radius of 4.9 nm. The apparent molecular weight and the frictional ratio of the agonist-receptor complex were computed to be 220,000 and 1.13, respectively. Dissociation of [3H]PAF from the radioligand-receptor complex was facilitated by Na+ and Li+, whereas K+ and Cs+ were ineffective. The guanine nucleotide, GTP, was also found to promote the dissociation in a manner that is additive with the effect of Na+, suggestive of the coupling of a guanine nucleotide binding protein to the solubilized PAF-receptor complex.
Chau
L Y
LY
Division of Eicosanoid Research, Institute of Biomedical Sciences, Taipei, Taiwan, China.
Jii
Y J
YJ
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D019204:GTP-Binding Proteins / Q000097:blood; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008565:Membrane Proteins / Q000097:blood*; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification*; D043562:Receptors, G-Protein-Coupled; D012964:Sodium / Q000494:pharmacology; D014316:Tritium
D008565:Membrane Proteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D014316:Tritium; D006160:Guanosine Triphosphate; D012964:Sodium; D019204:GTP-Binding Proteins