A method for assessing the binding of 3H labeled prostaglandin E1 ([3H]PGE1) to cell membranes has been developed and used to study the interaction of [3H]PGE1 with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [3H]PGE1 binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [3H]PGE1 binding activity with the presence or absence of PGE1 sensitive adenylate cyclase in several clones. In clone B82, a murine L cell, [3H]PGE1 binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 x 108M-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE1 (K(D)=30 nM) and kinetic analysis of [3H]PGE1 binding (k1=4 x 106 liters/mol/min, k-1 = 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clones N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE1 responsive adenylate cyclase, no specific [3H]PGE1 binding is detectable.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1976|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology