Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum

The Ca²⁺ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca²⁺ and surface membrane-bound Ca²⁺ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca²⁺ in the absence of ATP, 10–12 h are necessary for measurable amounts of Ca²⁺ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca²⁺ from ‘loaded’ vesicles only after this period of incubation. A fraction of Ca²⁺ of 50–60nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg²⁺ and K⁺ in the medium and is readily released by ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca²⁺ (30–40 nmol/mg protein) is rapidly released by X-537A. The results indicate that most of the Ca²⁺ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca²⁺ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.