Characterization of a Ca2+‐Dependent Guanylate Cyclase in the Excitable Ciliary Membrane from Paramecium

Abstract

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 μM and 36 μM, 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 (< 100 μM) and inhibited by high concentrations (> 100 μM) of calcium, half-maximal effects were obtained with 8 μM and 500 μM 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; K_m 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 μM, 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 EGTA 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/polassium action potential of Paramecium.