Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase

A strong connection with nucleotide activation of Ca2+ ATPase and phosphola mban inhibition has been found. Phospholamban decreases the number of activ atable Ca2+ ATPase without affecting substrate affinity or the ability of n ucleotide to serve its dual modulatory roles, i.e., catalytic and regulator y. Low concentrations of certain nucleotide mimetics, quercetin, tannin, an d ellagic acid, with structural similarity to adenine can unmask phospholam ban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approxima te to 0.3 mu M) and ellagic acid (EC50 approximate to 3 mu M) stimulated Ca 2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in iso lated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ ATPase was f ollowed by pronounced inhibiton at only slightly higher tannin concentratio ns (IC50 approximate to 3 mu M), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 mu M) than the EC 50. A complex relationship between compound, SR protein, and MgATP concentr ation is a major determining factor in the observed effects. Stimulation wa s only observed under conditions of phospholamban regulation, while the inh ibitory effects were observed in cardiac SR at micromolar Ca2+ and in skele tal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ ATPas e was identical to that observed with the anti phospholamban monoclonal ant ibody 1D11. Both compounds appear to relieve the Ca2+ ATPase from phosphola mban inhibition, thereby increasing the calcium sensitivity of the Ca2+ ATP ase like that observed with phosphorylation of phospholamban or treatment w ith monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a Linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibiti on of nucleotide activation. The data suggest that nucleotide activation of Ca2+ ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulati on. Clearly, this portion of Ca2+ ATPase needs further study to elucidate i ts role in phospholamban inhibition.