The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III.

Release of Ca2+ from the (Ca2+ + Mg2+)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca2+. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca2+ + Mg2+)-ATPase in the presence of Ca2+ causes the rapid phosphorylation of the enzyme at which time the bound Ca2+ becomes inaccessible to external EGTA. The release of Ca2+ from the (Ca2+ + Mg2+)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded form of the Ca2+-binding site which precedes the release of Ca2+ into the vesicle. The rate of Ca2+ accumulation by sarcoplasmic reticulum vesicles is increased by K+ (5-100 mM) and ATP (50-1000 microM) but the initial rate of Ca2+ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca2+ was not influenced by these concentrations of K+ and ATP.