Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.
Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP  receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors  eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D011494:Protein Kinases / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D000345:Affinity Labels; D008565:Membrane Proteins; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D011494:Protein Kinases