Solubilization of the [8-lysine]vasopressin receptor and adenylate cyclase from pig kidney plasma membranes.
Adenylate cyclase and the [8-lysine]vasopressin receptor were solubilized from pig kidney medulla membranes using the nonionic detergent Triton X-100. Optimal conditions for solubilization were under continuous stirring in a medium containing 0.5% (/v) Triton X-100, 100 mM Tris-HCl, pH 8, and 10 mM MgCl2. Both adenylate cyclase activity and [3H][8-lysine]vasopressin binding activity were recovered in a -26,000 X g supernatant of detergent-treated membranes. The yield of solubilized adenylate cyclase was nearly 100%. The soluble enzyme was no longer sensitive to antidiuretic hormone but was slightly activated by sodium fluoride. The affinity of the soluble receptor for [8-lysine]vasopresin was les than that of the membrane-bound receptor (mean apparent Km values, respectively 10(-7) M and 2 X 10(-8) M), however binding cooperativity was preserved. Hill coefficients were 1.42 for the soluble receptor and 1.50 for the membrane receptor. The soluble receptor discriminated as efficiently as did the membrane receptor between [8-lysine-a1vasopressin and oxytocin. The yield of spolubilized receptor was only 30% despite the fact that all binding activity had disappeared from the residual pellet of detergent-treated membranes. When the membranous receptors were occupied before solubilization and the latter was performed under conditions in which dissociation of the hormone-receptor comples is slow, i.e. at low temperature, 65% to 100% of the hormone-receptor complex was recovered in the soluble fraction. The soluble hormone-receptor complex partially dissociated on rewarming whereas the free hormone concentration was kept unchanged in the medium. The residual binding capacity, which was 30% of the initial value, was identical with that determined when the receptor was solubilized in free form before incubation with labeled hormone. It was concluded that (a) solubilization of the receptor molecules was complete, (b) during solubilization two forms of the receptor appear, of which only one is accessible to the hormone, (c) occupancy of the receptor by the hormone prevented the formation of the nonaccessible form, and (d) some component or components of the soluble fraction might be responsible for the loss in apparent affinity.
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007679:Kidney Medulla / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008236:Lypressin / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism; D011092:Polyethylene Glycols; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011956:Receptors, Cell Surface; D012995:Solubility; D013552:Swine; D014667:Vasopressins / Q000031:analogs & derivatives*
D011506:Proteins; D011956:Receptors, Cell Surface; D014667:Vasopressins; D011092:Polyethylene Glycols; D010121:Oxytocin; D008236:Lypressin; D000262:Adenylyl Cyclases