Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium.
The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D002923:Cilia / Q000201:enzymology*; D006162:Guanylate Cyclase / Q000378:metabolism*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008674:Metals, Rare Earth / Q000494:pharmacology; D010247:Paramecium / Q000201:enzymology*; D013696:Temperature
D008674:Metals, Rare Earth; D007811:Lanthanum; D006162:Guanylate Cyclase; D002118:Calcium