Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout

Abstract

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membranes of freshwater-adapted trout by measuring the conversion of [α- 32P]ATP into [α- 32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20°. The K m for ATP was 0.5 m M in the presence or absence of the stimulators. The presence of 10 −5 M guanosine-5′-triphosphate and 4 × 10 −3 M MgCl 2 (2.41 × 10 −3 M free Mg 2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca 2+ was inhibitory. IC 50 for CaCl 2 was 5 × 10 −4 M (10 −7 M free Ca 2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400–450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE 1 10 −5 M (50% increase over basal activity) or by glucagon 5.7 × 10 −10 M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10 −2 M.