JBC, Vol. 250, Issue 19, 7586-7592, Oct, 1975
Solubilization of calcitonin-responsive renal cortical adenylate cyclase
S. F. Queener, J. W. Fleming and N. H. Bell
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 mum 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 1mM dithiothreitol and 5mM 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 muM calcitonin. Binding of
125I-labeled 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 muM porcine calcitonin and
maximal effect at a concentration between 33 and 66 muM porcine calcitonin.
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