CHARACTERIZATION OF CALCIUM-ACTIVATED ADENOSINE TRIPHOSPHATASE IN THE CORAL, GALAXEA FASCICULARIS

Abstract

Ca2+- activated ATPase activity was detected in the, coral Galaxea fascicularis. Studies on the activities of alkaline phosphatase, glucose-6-phosphatase and cytochrome oxidase indicated that the Ca2+- activated ATP hydrolysis was located in the plasma membrane enriched heavy microsomal fraction of G. fascicularis. Enzyme activity was dependent on Ca2+ and ATP concentrations. Two components of Ca2+- ATPase with differing affinities for Ca2+ were obtained. Woolf-Augustinsson-Hofstee transformation of data yielded a Vmax and Km of 0.048 umol Pi/mg protein per min and 0.002 mM respectively for the high-affinity Ca2+- ATPase. The Vmax and Km for the low-affinity component were 0.75 umol Pi / mg protein per min and 0.16 mM respectively. High ATP concentrations decreased enzyme activities of both components. The activity of the high-affinity Ca2+- induced ATP hydrolysis was inhibited by the calmodulin antagonists calmidazolium ( 80.4% ) and chlorpromazine ( 51.1% ) while the low-affinity component was unaffected . Substrate specificity of enzyme activities for ATP alone could not be demonstrated for both high- and low-affinity Ca2+ -stimulated ATPase possibly because enzyme sample consisted of other contaminatinq membrane fractions. Both Ca2+ -ATPase activities in G. fascicularis were not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea ( DCMU ), dinitrophenol, Diamox, ethacrynic acid, potassium cyanide, sodium fluoride, iodoacetate, ouabain, oligomycin B, Diamox and L-phenylalanine. Low-affinity Ca2+- ATPase activity was inhibited 20.0% and 14.4%, by theophylline and sodium azide respectively. Mersalyl acid and p-chloromercuribenzoate inhibited both high- and low-affinity components indicating the enzymes' requirement for sulphydryl groups. High-affinity Ca2+- ATPase activity was totally inhibited by ruthenium red while activity of the low-affinity component was inhibited by 63.7%. Both high- and low-affinity Ca2+- stimulated ATPase activities were optimal at pH 7.0 and at 45°c. Strontium ion was a competitive inhibitor of both high- and low-affinity Ca2+- ATPase and cannot substitute Ca2+ in activating the ATP hydrolysis. Ki for the high- and low-affinity components were 0.0085 and 2.056 mM respectively. Other ATPases were also detected in the plasma membrane enriched fraction with the exception of Sr2+- stimulated ATPase. The high-affinity calmodulin-dependent Ca2+- ATPase in G. fascicularis possess many properties characteristic of plasma membrane Ca2+- transport ATPase in eukaryotic systems. Its affinity for micromolar concentrations of Ca2+ makes it a more likely candidate than the low-affinity component for Ca2+- transport and calcification in corals. Results from this investigation strongly suggests that the transport of Sr2+ and Ca2+ involve different mechanisms.