Maximal calcium extrusion capacity and stoichiometry of the human red cell calcium pump.

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Abstract
1. Thee uphill calcium efflux through calcium-saturated pumps in intact red cells was investigated with the aid of a new method, in initial conditions of uniform ionophore A23187-induced calcium distribution among the cells. The method is based on findings by Tiffert, Garcia-Sancho and Lew (1984) which show that cobalt can suddenly arrest passive calcium transport by the ionophore and expose, without noticeable interference, uphill calcium extrusion by the pump. The results comprise methodological aspects and questions concerning interactions between inner pump sites, ATP and Ca2+, and the calcium:ATP stoichiometry of the calcium-saturated pump. 2. Ionophore-induced calcium influx was set to be far in excess of the maximal calcium pump capacity. This secured a uniform calcium distribution among the cells, and Ca2+ equilibation by 2 min or less of calcium permeabilization. Cobalt was added between 15 s and 5 min after ionophore addition. The calcium and ATP content of the cells was followed during ionophore-induced influx and cobalt-exposed efflux. 3. The external cobalt concentrations required to block completely ionophore-mediated calcium transport were similar or only marginally higher than those of calcium. 4. The reproducibility of independent cobalt-exposed calcium efflux measurements from single blood samples was within an 8% range. 5. During cobalt-exposed calcium efflux, the calcium content of subpopulations of cells, with and without active Ca2+-sensitive K+ channels, investigated by postincubation of samples in low K+, thiocyanate (SCN-) media (modified from Garcia-Sancho and Lew, 1988a), was similar. This is consistent with the maintenance of uniform calcium distribution among the cells during uphill calcium extrusion. 6. Cobalt-exposed calcium efflux was similar in the interval from 15 s to 5 min after calcium permeabilization although cell ATP levels had fallen by over 50% in that period. Therefore, cell ATP concentrations within the physiological range do not seem to be regulatory for calcium-saturated pumps in the intact red cell. 7. All cobalt-exposed calcium efflux curves were linear in time, at least until total cell calcium contents reached levels below 100 .mu.mol/l cells. This suggest that internal calcium is not inhibitory for calcium-saturated efflux in intact cells in the 0.1-1 mmol/l cells range. 8. The cobalt-exposed calcium fluxes were in the range from 4 to 24 mmol/l cells.h) for fresh cells and from 10 to 18 mmol/(l cells.h) for samples from the Blood Bank. This variability was documented for blood samples from different donors and also for the same donor in samples obtained on different days. This variability can be attributed to unknown factors that control the fraction of active pumps, or the turnover of calcium-saturated pumps, in intact red cells. 9. Simultaneous measurements of cobalt-exposed calcium efflux and of the calcium-dependent ATP fall following calcium permeabilization of fresh red cell incubuated in the absence of substrates, were used to estimate the calcium:ATP stoichiometry of the calcium-saturated pump. The scatter of the experimental points was large but all fell within the region predicted for a 1:1 stoichiometry once allowance was made for the ATP-buffering effects of adenylate kinase.