A NEW METHOD FOR MONITORING THE KINETICS OF CALCIUM-BINDING TO THE SARCOPLASMIC-RETICULUM CA-2+-ATPASE EMPLOYING THE FLASH-PHOTOLYSIS OF CAGED-CALCIUM

The kinetics of Ca2+ binding to the high-affinity sites of the sarcoplasmic reticulum (SR) Ca2+-ATPase were directly investigated by continuously monitoring the extravesicular calcium concentration via the metallochromic indicator Arsenazo III following the release of Ca2+ from a photolabile caged-calcium molecule, 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N′,N′-tetrakis [(oxycarbonyl)methyl]-1,2-ethanediamine DM-nitrophen), utilizing a pulsed Nd:YAG laser for photolysis. The nature of the binding kinetics is at least biphasic over the first 400 ms for vesicular dispersions of SR. The stoichiometry for calcium binding expressed as Ca:E1 ′ P has been calculated to be ′ 1.4:1 for the pure SR preparation under the reaction conditions employed. © 1990.