Asymmetric Distribution of Calcium Binding Sites of Sarcoplasmic Reticulum Fragments

Abstract

The calcium binding characteristics of sarcoplasmic reticulum fragments (SRF) of rabbit skeletal muscle were studied in vitro by a method discriminating the outside and inside of the vesicles. After equilibration with ⁴⁵Ca under physiological salt conditions, the total amount of calcium bound on both sides was determined by analyzing the calcium concentration of the SR suspension before and after ultracentrifugal separation. The amount of calcium bound on the inside and free calcium in the inner water space was determined by analyzing the calcium content of SRF after filtration through a Millipore filter. The inner space was determined in terms of the inulin exclusion volume. The binding data were analyzed by means of a Scatchard plot. It was found that there are two kinds of calcium binding sites on the outside and four kinds of sites on the inside of the vesicles. The numbers of sites (nmol/mg SRF protein) and the dissociation constants (M) (in parentheses) are as follows. Outside: α₁, 11.9 (8.4×10⁻⁷); and α₂, 62(4.1 ×10⁻³) Inside: β₀, 1.4(2.7×10⁻⁷); β₁, 2.1 (4.7×10⁻⁶); β₂, 44.8 (1.05×10⁻³); and β₃, 153 (3.8×10⁻²). By comparing the dissociation constants and the numbers of these sites with those of purified proteins of sarcoplasmic reticulum, the arrangement of main protein components was determined. The calcium translocating site of the calcium pump protein (corresponding to α₁) faced outside. Calcium binding sites of high affinity calcium binding protein (β₁) and calsequestrin (β₂) were inside the vesicles.