Solubilization of functional calcitonin receptors

Biochem J. 1988 Jul 15;253(2):505-10. doi: 10.1042/bj2530505.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azides
  • Binding Sites
  • Binding, Competitive
  • Calcitonin / analogs & derivatives
  • Calcitonin / metabolism
  • Cell Membrane / metabolism
  • Cholic Acids
  • Detergents
  • Electrophoresis, Polyacrylamide Gel
  • Female
  • Humans
  • Placenta / metabolism*
  • Pregnancy
  • Receptors, Calcitonin
  • Receptors, Cell Surface / isolation & purification*
  • Receptors, Cell Surface / metabolism
  • Solubility

Substances

  • Azides
  • Cholic Acids
  • Detergents
  • Receptors, Calcitonin
  • Receptors, Cell Surface
  • calcitonin, (4-azidobenzoyl)-Arg(11,18)-Lys(14)-
  • Calcitonin
  • 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate