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Abstract:
Calcium fluxes across the membranes of the sarcoplasmic reticulum vesicles supported by ATP and by GTP were analyzed with respect to the effects of the reaction products nucleoside diphosphate, Pi and intravesicular calcium. Calcium fluxes and hydrolysis of nucleoside triphosphate (NTP) were determined after cessation of net uptake of calcium and during phosphate‐supplemented net storage of calcium. After net uptake of calcium has ceased the calcium transport enzyme splits ATP and GTP with only 10% of its optimal activity, due to the inhibiting effect of high intravesicular calcium concentrations. The same maximal calcium load of 80 nmol/mg is obtained with ATP and GTP but maintained at very different rates of calcium exchange which correspond to the suppressed rates of ATP and GTP splitting. Thus, calcium influx is coupled to ATP and GTP splitting with nearly the same ratio of 0.6–0.8. The calcium‐dependent NTPase activity is less sensitive to the inhibiting effect of both nucleoside diphosphates at high concentrations than it is at low concentrations of intravesicular calcium. In contrast to the NTPase activity displayed by calcium‐filled vesicles the exchange between internal and external calcium is markedly stimulated by the addition of ADP and GDP. Evidently, at zero net uptake of calcium, calcium translocation is not coupled to phosphate liberation. In the absence of nucleoside diphosphates the addition of inorganic phosphate causes a permanently proceeding net uptake of calcium and enhances the calcium‐dependent NTPase activity. The simultaneously occurring calcium efflux, however, remains unaffected by phosphate. The resulting increment of calcium influx is matched by the increment of NTP hydrolysis, thus leaving the ratio calcium influx NTPase activity unchanged. In the presence of nucleoside diphosphates, however, the addition of phosphate accelerates calcium influx as well as calcium efflux. The rates of the resulting net uptake of calcium only reach 2% or 10%, respectively, of the rate of calcium influx supported by ATP or GTP. The rates of calcium movement together with the known rates of unidirectional calcium uptake and release are discussed in relation to the activity of the various phosphate‐transfer reactions in the transport cycle.
