Rapid kinetic studies of active Ca²⁺ transport in sarcoplasmic reticulum

Summary

A method is reported for the rapid and continuous monitoring of active Ca²⁺ transport events occurring in isolated skeletal sarcoplasmic reticulum (SR). The method is based on the quantitative evaluation of increases in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS⁻), resulting from active transport. The method, which has a time resolution of 20 msec, was applied to the kinetics of Ca²⁺ transport by a Ca²⁺-ATPase-rich SR fraction and the effects and loci of action of Mg²⁺ and monovalent cations (M ⁺) were investigated. The turnover number of the enzyme and its ability to establish gradients were investigated in the absence of the complicating effects of precipitating anions. The results are explicable in terms of the model of Kanazawa et al. (Kanazawa, T., Yamada, S., Yamamoto, T., Tonomura, Y., 1971,J. Biochem. (Tokyo) 70:95) and are difficult to reconcile with models in which the enzyme is considered to be electrogenic. The major observations of the study are as follows:

• 1) Active uptake of 29 μM free Ca²⁺ in the presence of 5mm KCl, initiated by the addition of 10⁻⁴ m Mg²⁺ and 2×10⁻⁴ m ATP, occurs with at _1/2 of ca. 9 sec. The process results in an internal free Ca²⁺ concentration of 13mm.

• 2) Preincubation with 50mm KCl and 5mm MgCl₂, followed by initiation of active uptake by the addition of ATP to give a final concentration of ca. 2.5mm Mg²⁺ and ca. 2.5mm MgATP, gave faster and larger uptakes. Thet _1/2 for the reaction was ca. 600 msec and the internal free Ca²⁺ concentration was 70±20mm. The turnover number of 7.1±0.8 sec⁻¹ was calculated for the enzyme at mid-reaction under the assumption of a stoichiometry of 2 Ca²⁺ per cycle.

• 3) The accelerative effects of Mg²⁺ andM ⁺ on the rate of transport were investigated. Experiments in which the cations were added to or omitted from the incubation medium showed that the presence of both classes of activator in the internal aqueous space was necessary for maximal activation of the transport system. The concentration dependencies of these effects were investigated. Analysis shows that the monovalent cation effect is probably based on the countertransport according to the model of Kanazawa et al. (1971) while the Mg²⁺ effect referable to the inside surface is primarily catalytic. No Mg²⁺ counter-transport could be demonstrated under conditions in which the internal monovalent cation concentration was adequate.

• 4) Under conditions in which the Mg²⁺ concentration is adequate for stimulation but theM ⁺ concentration is not (and vice versa), the active uptake can be resolved into two phases. The rapid phase is complete within the first 50 msec and corresponds to 0.26–2.06 Ca²⁺ released to the internal phase per Ca²⁺-ATPase. These results correspond closely to those of published studies measuring the rate at which Ca²⁺ becomes inaccessible to the external solution. The comparison shows that Ca²⁺ is released to the internal aqueous phase almost as rapidly as it becomes inaccessible to the outside phase. Analysis of the concentration dependencies shows that K⁺/Ca²⁺ or Mg²⁺/Ca²⁺ competition for occupation of the inwardly-oriented translocator (of the phosphorylated enzymes) is involved in the fast phase of Ca²⁺ release. When the internal concentrations of both K⁺ and Mg²⁺ are adequate, the slow phase is speeded up to such an extent that the first partial turnover can no longer be kinetically isolated from the subsequent turnovers. Under these conditions, the rate of enzyme dephosphorylation, the binding of K⁺ to the translocator, and its return to an outward orientation are no longer rate limiting. The rate constant for the outward-to-inward reorientation of this translocator is ca. 13.8 sec⁻¹. The average turnover number for the first several turnovers, obtained under conditions of maximal stimulation, is ca. 7.1. The latter value was somewhat influenced by trans-inhibition by internal Ca²⁺. It is concluded that this outward-to-inward transition of the Ca²⁺-laden translocator is rate-limiting to the first turnover and that the rate of the inward-to-outward transition of the K⁺-laden translocator becomes limiting in the final phases of the transport process.

• 5) Two major lines of evidence against electrogenic models of pump function were the stimulatory effect of internal K⁺ on the transport reaction and the lack of a stimulatory effect as an inwardly-directed Cl⁻ gradient. Also the mechanism of the reponse of the KCl impermeable vesicles to valinomycin was investigated. Those findings also run counter to the expectations of electrogenic pump mechanisms.