The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 mu M, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+](lim), whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+](lim) was decreased 70% to 80% by histidine, imidazole and cysteine. The K-0.5 value for histidine was 3 mM, suggesting that residual [Ca2+](lim) fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a. corrected value for [Ca2+](lim) of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+](lim) remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+](lim) levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+](lim) was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+](lim) was estimated by the unidirectional flux of Ca-45(2+), and directly, following depletion of ATP, by measuring release of Ca-40(2+), and was 0.02% of V-max. Constant infusion of CaCl2 at [Ca2+](lim) resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were V-max = 853 nmol/min per mg, K-0.5(Ca) = 279 nM, and n(H(Ca)) = 1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+](lim) in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways. (C) 1999 Elsevier Science B.V. All rights reserved.
