Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes.
A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.
Chau
L Y
LY
Division of Eicosanoid and Lipid Mediator Research, Institute of Biomedical Sciences, Taipei, Taiwan, Republic of China.
Tsai
Y M
YM
Cheng
J R
JR
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D007700:Kinetics; D010972:Platelet Activating Factor / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled
D000345:Affinity Labels; D001386:Azides; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; C060159:1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn-glycero-3-phosphocholine