Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets.
Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.
D000262:Adenylyl Cyclases / Q000032:analysis; D000527:Alprostadil / Q000378:metabolism*; D001665:Binding Sites; D001792:Blood Platelets / Q000032:analysis*; D002118:Calcium / Q000494:pharmacology; D004789:Enzyme Activation; D011464:Epoprostenol / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000302:isolation & purification*; D018078:Receptors, Prostaglandin E
D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D011464:Epoprostenol; D000262:Adenylyl Cyclases; D000527:Alprostadil; D008274:Magnesium; D002118:Calcium