Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged ATP

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Abstract
The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of [rabbit] sarcoplasmic reticulum were determined with a time resolution of .apprx. 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyle adenosine 5''-triphosphate utilizing a frequency-doubled ruber laser and measured with 2 different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2.degree. to 26.degree. C. The Ca2+ ionophore A23187 [calcimycin] was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E .apprx. P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least 2 phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 .+-. 10 s-1 at 23.degree.-26.degree. C, an activation energy of 16 .+-. 1 kcal mol-1, and a .DELTA.S* of .apprx. 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E .apprx. P, with stoichiometry of .apprx. 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 .+-. 0.09 s-1 at 25.degree.-26.degree. C, an activation energy of 22 .+-. 1 kcal mol-1, and .DELTA.S* of .apprx. 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of .apprx. 2 mol of Ca2+/mol of ATP hydrolyzed.