Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout.
Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membrane of freshwater-adapted trout by measuring the conversion of [alpha-32P]ATP into [alpha-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 degrees. The Km for ATP was 0.5 mM in the presence or absence of the stimulators. The presence of 10(-5) M guanosine-5'-triphosphate and 4 X 10(-3) M MgCl2 (2.41 X 10(-3) M free Mg2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca2+ was inhibitory. IC50 for CaCl2 was 5 X 10(-4) M (10(-7) M free Ca2+) 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 PGE1 10(-5) M (50% increase over basal activity) or by glucagon 5.7 X 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.
D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology; D005880:Gills / Q000187:drug effects / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D014176:Protein Biosynthesis; D012487:Salmonidae / Q000502:physiology*; D012969:Sodium Fluoride / Q000494:pharmacology; D013696:Temperature; D014337:Trout / Q000502:physiology*
D002413:Cations, Divalent; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012969:Sodium Fluoride; D000262:Adenylyl Cyclases; D007545:Isoproterenol