Solubilization of calcitonin responsive renal cortical adenylate cyclase

S. F. Queener, J. W. Fleming, N. H. Bell

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

Purification of pork renal cortex membranes yielded a particulate adenylate cyclase retaining good sensitivity to stimulation by parathyroid hormone and glucagon and a modest but significant response to porcine calcitonin. Treatment of this partially purified membrane fraction with 0.5% Lubrol PX and 5 mM NaF released adenylate cyclase activity into a fraction which was not sedimented by centrifugation for 20 min at 37,000 x g or for 2 hours at 100,000 x g and passed through a Millipore filter (0.22 μm pore). This solubilized adenylate cyclase was stimulated by porcine calcitonin and NaF but not by parathyroid hormone or glucagon. On gel filtration (Sephadex G 200) in the presence of 1 mM dithiothreitol and 5 mM NaF, the major portion of the adenylate cyclase activity eluted with the void volume of the column and showed 2.0 fold stimulation with 10 μM calcitonin. Binding of 125I labelled porcine calcitonin was demonstrated in the 37,000 x g and the 100,000 x g supernatants. From 74 to 86% of the observed binding could be blocked by the addition of unlabeled porcine calcitonin to the reaction mixture. Addition of salmon calcitonin, parathyroid hormone, or glucagon blocked only 12 to 18% of the binding. The dose response curves for inhibition of binding of iodinated calcitonin by unlabeled calcitonin and the activation of adenylate cyclase by the hormone each showed 50% maximal effect at a concentration between 4.5 and 8 μM porcine calcitonin and maximal effect at a concentration between 33 and 66 μM porcine calcitonin.

Original languageEnglish (US)
Pages (from-to)7586-7592
Number of pages7
JournalJournal of Biological Chemistry
Volume250
Issue number19
StatePublished - Dec 1 1975
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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