Analysis of sodium fluoride enhancement of calcitonin stimulation of renal cortical adenylate cyclase.

NaF (5 mu) was previously shown to protect calcitonin-responsive adenylate cyclase activity in partially purified membranes from pork renal cortex during solubilization of the enzyme with Lubrol PX (Queener, S. F., Fleming, J. W., and Bell, N. H. (1975) J. Biol. Chem. 250, 7586-7592). NaF has now been found to directly influence the activity of the membrane-bound calcitonin-responsive adenylate cyclase. At low concentrations of NaF causing no direct stimulation of adenylate cyclase activity, NaF produced a synergistic increase with calcitonin but not with parathyroid hormone or glucagon. At maximally stimulating concentrations of NaF, calcitonin produced a further increase in enzyme activity. NaF alone increased the maximal velocity (I’,,) and did not affect the apparent K, of the enzyme for ATP, whereas calcitonin alone did not alter the V,, but lowered the K, for ATP. Concentrations of NaF too low to directly alter either K,,, or V,, significantly increased V,, and decreased K,,, when added with calcitonin. De Ha&n plots (De Ha&, C. (1974) J. Biol. Chem. 249, 2756-2762) of adenylate cyclase activation by calcitonin with and without NaF indicated that NaF increased the maximal response of the enzyme to calcitonin without affecting the affinity of the enzyme for calcitonin. These effects of NaF could not be attributed to alterations in the activity of phosphodiesterase in these membranes. In addition, NaF had no effect on the slope of the Hill plot for activation by calcitonin and, likewise, calcitonin had no effect on the slope of the Hill plot for activation by NaF. NaF did not alter binding of ‘251-calcitonin to renal cortical membranes or the degradation of calcitonin as determined by chromatoelectrophoresis and radioimmunoassay. NaF was shown to increase the heat lability of the enzyme. It is concluded (a) that NaF at low concentrations acts to enhance responsiveness of renal cortical adenylate cyclase to calcitonin without altering binding or degradation of the hormone and (b) that NaF at high concentrations directly stimulates the catalytic component of the enzyme independent of any action of the hormone. These results suggest that NaF acts at different sites to influence the calcitonin-responsive adenylate cyclase system of pork renal cortex.