Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

Abstract

The GTP-driven component of Ca(2+) uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca(2+)-translocating ATPase and assess its utility as a probe for this transport system. Uptake of (45)Ca(2+) in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca(2+) concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca(2+)-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of (45)Ca(2+)-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven (45)Ca(2+) uptake represents the capacity of the plasma membrane Ca(2+)-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [gamma-(32)P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca(2+)-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca(2+)-ATPases present in animal cells.