Reversible calcitonin binding to solubilized sheep brain binding sites

P. M. Sexton, H. G. Schneider, C. S. D'Santos, F. A.O. Mendelsohn, B. E. Kemp, J. M. Moseley, T. J. Martin, D. M. Findlay

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In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of 125 I-labelled salmon CT ( 125 I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with β-octyl glucoside. 125 I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 ± 0.5 nM and a maximum binding site concentration of 6.2 ± 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems. Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mM. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 125 I-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40% and 46% dissociation after 2 h at 4°C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125 I-[Arg 11,18 ,4-azidobenzoyl-Lys 14 ]-sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of M(r) ~72,000 in both solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified, one of M(r) ~71,000 and another of M(r) ~88,000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues.

Original languageEnglish
Pages (from-to)179-184
Number of pages6
JournalBiochemical Journal
Issue number1
Publication statusPublished - 1 Jan 1991
Externally publishedYes

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