Pierce D H, Scarpa A, Topp M R, Blasie J K
Biochemistry. 1983 Nov 8;22(23):5254-61. doi: 10.1021/bi00292a003.
The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two 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 to 26 degrees C. The Ca2+ ionophore A23187 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 approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two 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 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 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 approximately P, with a stoichiometry of approximately 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 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.
利用倍频红宝石激光通过对P3 - 1 - (2 - 硝基苯基) - 乙基腺苷5'-三磷酸进行闪光光解添加ATP来启动肌浆网囊泡分散体对ATP诱导的Ca2+摄取动力学研究，其时间分辨率约为10毫秒，具体取决于温度。Ca2+摄取通过两种不同的检测系统进行测量，这两种系统跟踪对细胞外[Ca2+]变化敏感的金属显色指示剂偶氮胂III的吸光度变化。研究的温度范围是 - 2至26摄氏度。Ca2+离子载体A23187用于区分与跨膜Ca2+梯度形成相关的Ca2+摄取动力学特征。采用淬灭流动技术独立测定酸稳定的磷酸化酶中间体E≈P。Ca2+摄取至少有两个阶段，一个快速初始阶段和一个缓慢阶段。快速阶段表现出假一级动力学，在23 - 26摄氏度下的比速率常数为64±10 s-1，活化能为16±1 kcal mol-1，ΔS约为5 cal deg-1 mol-1，对Ca2+离子载体的存在不敏感，并且与磷酸化酶E≈P的形成同时发生，化学计量比约为2 mol Ca2+/mol磷酸化酶中间体。缓慢阶段也表现出假一级动力学，在25 - 26摄氏度下的比速率常数为0.60±0.09 s-1，活化能为22±1 kcal mol-1，ΔS约为16 cal deg-1 mol-1，受到Ca2+离子载体的抑制，化学计量比约为2 mol Ca2+/mol水解的ATP。
