Allosteric Inhibition by Alkali Ions of the Ca2+ Uptake and Adenosine Triphosphatase Activity of Skeletal Muscle Microsomes

Abstract

Abstract The Ca2+ uptake and the calcium-activated adenosine triphosphatase activity of skeletal muscle microsomes are inhibited by alkali ions in assay media containing ATP concentrations ranging from 10-6 to 10-5 m. No inhibition is observed at ATP concentrations higher than 10-4 m. The pattern of inhibition of Ca2+ uptake by different alkali ions varies depending on the ATP concentration. For 2 µm ATP, the sequence of inhibition is Li+ g Na+ g K+ and for 10 µm ATP is Na+ g Li+ ≥ K+. The calcium-activated ATPase is inhibited according to the same pattern, Na+ g Li+ ≥ K+, at both 2 and 10 µm ATP. The dependence of the initial rate of Ca2+ uptake on the ATP concentration displays hyperbolic saturation curves in assay media containing 20 mm KCl, 120 mm KCl, or 120 mm NaCl. Na+ and K+ inhibit both the Ca2+ uptake and the calcium-activated ATP-ase activity to the same extent. In contrast, sigmoidal saturation curves for Ca2+ uptake and hyperbolic saturation curves for the calcium-activated ATPase are obtained in media containing 120 mm LiCl. When the free Ca2+ concentration is varied from 1.0 to 7.1 µm in media containing 10 µm ATP and 20 mm KCl, 120 mm KCl, or 120 mm NaCl, both the initial rate of Ca2+ uptake and the calcium-activated ATPase activity increase hyperbolically. Lowering the ATP concentration to 2 µm results in sigmoidal saturation curves for Ca2+ uptake and hyperbolic saturation curves for the calcium-activated ATPase activity. The Ca2+ uptake in media containing 120 mm LiCl is a sigmoidal function of the Ca2+ concentration both at ATP concentrations of 2 µm and 10 µm. However, the calcium-activated ATPase always shows a hyperbolic dependence on the Ca2+ concentration. The effect of alkali ions on Ca2+ influx and efflux through the skeletal muscle microsomes membrane was studied with the use of 2 µm ATP. Both the rate of Ca2+ efflux and of active transport are inhibited by Na+ and Li+. In media containing 120 mm of either NaCl or LiCl, the equilibrium between active transport and Ca2+ efflux is reached at Ca2+ concentrations higher than 0.5 µm. The rate of Ca2+ exchange between the interior of the vesicles and the assay medium is essentially the same in the presence or absence of 120 mm of either NaCl or LiCl. The data presented suggest that: (a) acetyl phosphate and ATP are both substrates of a common active site for the Ca2+ transport; and (b) the binding of ATP to the Ca2+ carrier system promotes conformational changes which result in modification of its affinity for Ca2+.