Prostaglandin F2alpha receptors in bovine corpus luteum cell membranes. Effect of enzymes and protein reagents.
Various enzymes and protein reagents inhibited [3H]prostaglandin F2alpha binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components. Preincubation of membranes with phospholipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and N-ethylmaleimide resulted in moderate to drastic inhibitions of [3H]prostaglandin F2alpha binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for N-ethylmaleimide), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca2+ was not required for its inhibition of [3H]prostaglandin F2alpha binding. Protein reagents inhibition of binding was differently influenced by added Ca2+ i.e., loss of binding increased with some (N-ethylmaleimide), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca2+ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups. Treatment of membranes with trypsin and N-ethylmaleimide selectively abolished high affinity prostaglandin F2alpha receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzene and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F2alpha receptors or by severely reducing their affinities. The occupancy of receptors by prostaglandin F2alpha afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.
D000812:Anhydrides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003338:Corpus Luteum / Q000187:drug effects / Q000378:metabolism*; D004139:Dinitrofluorobenzene / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D009439:Neuraminidase / Q000494:pharmacology*; D010740:Phospholipases / Q000494:pharmacology*; D011402:Pronase / Q000494:pharmacology*; D011460:Prostaglandins F / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011955:Receptors, Drug / Q000187:drug effects*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013774:Tetranitromethane / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000812:Anhydrides; D011460:Prostaglandins F; D011506:Proteins; D011955:Receptors, Drug; D013439:Sulfhydryl Reagents; D004533:Egtazic Acid; D004139:Dinitrofluorobenzene; D010740:Phospholipases; D009439:Neuraminidase; D014357:Trypsin; D011402:Pronase; D013774:Tetranitromethane; D005033:Ethylmaleimide; D002118:Calcium