Solubilization of functional calcitonin receptors.
Calcitonin (CT) binding activity has been extracted from a membrane fraction of human placenta using the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid (Chaps). Approximately two-thirds of the available binding sites were extracted using 5 mM-Chaps. The binding characteristics of 125I-labelled salmon CT(125I-sCT) to the solubilized extract were similar to those obtained previously with placental membranes and other targets such as osteoclasts, renal cells and certain human cancer cell lines. 125I-sCT binding was saturable (Bmax. 75 +/- 6 fmol/mg of protein, n = 3) and Scatchard analysis revealed a single class of high-affinity binding sites (Kd 165 +/- 28 pM, n = 3). In competitive-binding studies, various species-specific CTs and CT analogues showed the same rank order of potencies as seen in CT bioassays and several unrelated peptides did not compete at high doses. A biologically active CT analogue, [Arg11,18, Lys14]sCT, derivatized with the photoreactive phenylazide cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate, was used to identify receptor components of Mr approximately 88,000 and approximately 71,000 in both particulate placental membranes and the solubilized extract. Receptor components of Mr 85-90,000 have been identified in other CT target cells previously using chemical- and photoaffinity-labelling techniques. These results demonstrate the first successful solubilization of the CT receptor in a form which purification.
Nicholson
G C
GC
Department of Medicine, University of Melbourne, Repatriation General Hospital, Heidelberg, Victoria, Australia.
D'Santos
C S
CS
Evans
T
T
Moseley
J M
JM
Kemp
B E
BE
Martin
T J
TJ
D001386:Azides; D001665:Binding Sites; D001667:Binding, Competitive; D002116:Calcitonin / Q000031:analogs & derivatives / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D001386:Azides; D002793:Cholic Acids; D003902:Detergents; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; C057407:calcitonin, (4-azidobenzoyl)-Arg(11,18)-Lys(14)-; D002116:Calcitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate