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When alpha,beta-methylene ADP (alpha,beta-CH2-ADP) is added to the phosphorylated calcium ATPase of sarcoplasmic reticulum with Ca(2+)-bound, Ca2.E approximately P.Mg, alpha,beta-methylene ATP is not synthesized (5 mM MgCl2, 100 mM KCl, pH 7.0, 25 degrees C). Similarly, adenosine 5'-O-(2-thiotriphosphate) is not synthesized from reaction of the phosphoenzyme with adenosine 5'-O-(2-thiodiphosphate), ADP beta S. In contrast, ATP is formed rapidly and reversibly from the reaction of the phosphoenzyme with ADP. Both ADP analogs are competitive inhibitors for the binding of ADP to the phosphoenzyme with KADPS = 0.45 mM: alpha,beta-CH2-ADP and ADP beta S bind to the phosphoenzyme with K alpha,beta-CH2-ADPS = 0.92 mM and KADP beta SS = 0.05 mM, respectively. We conclude that phosphoryl transfer from the phosphoenzyme to alpha,beta-CH2-ADP is kinetically blocked, although it is thermodynamically favorable. The rate acceleration of > 10(5) for phosphoryl transfer from Ca2.E approximately P.Mg to ADP compared to alpha,beta-CH2-ADP can be attributed to the differences in both the structure and the net charge of ADP compared with alpha,beta-CH2-ADP at pH 7.0. Phosphoryl transfer from the phosphoenzyme to ADP beta S is thermodynamically so unfavorable that we cannot determine whether the transition state is also unfavorable.

The calcium ATPase of sarcoplasmic reticulum reacts with inorganic phosphate (Pi) to form phosphoenzyme that can bind two Ca2+ ions from the lumen of intact vesicles. Therefore, as the concentration of lumenal Ca2+ is increased, the concentration of phosphoenzyme at equilibrium increases; however, it levels off at lower maximal concentrations with decreasing concentrations of Pi. This requires that two Ca2+ ions can bind to lumenal binding sites of both the phosphoenzyme and the unphosphorylated enzyme. If lumenal Ca2+ could bind only to the phosphoenzyme, saturating concentrations of lumenal Ca2+ would drive phosphoenzyme formation to completion even at low concentrations of Pi. Phosphorylation is inhibited by cytoplasmic Ca2+ with K0.5 = 2.1 and 4 microM in the absence and in the presence of 40 mM lumenal Ca2+, respectively. K0.5 = 4 microM is much lower than K0.5 = 70 microM, which is expected if lumenal Ca2+ could bind only to the phosphoenzyme. Occupancy of the lumenal sites on the unphosphorylated enzyme by Ca2+ does not significantly change the rate constants of kp = 220 s-1 for phosphorylation by ATP, kCa = 90 s-1 for dissociation of Ca2+, and kMg = 50 s-1 for dissociation of Mg2+. We conclude that the calcium ATPase has two low-affinity lumenal Ca(2+)-binding sites that are independent of the high-affinity cytoplasmic Ca(2+)-binding sites. The results are consistent with a mechanism of Ca2+ transport in which phosphorylation of the enzyme by ATP drives the translocation of two Ca2+ ions from the high-affinity to the low-affinity sites.

The calcium-transporting ATPase of sarcoplasmic reticulum is known to bind two Ca2+ ions from the cytoplasm to the free enzyme and two Ca2+ ions from the lumen to the phosphoenzyme. The concentration of phosphoenzyme formed at equilibrium from Pi and Mg2+ increases with increasing concentration of calcium in the lumen, which binds to the phosphoenzyme to form Ca2.E approximately P.Mg. However, at subsaturating concentrations of Mg2+ increasing the concentration of lumenal Ca2+ does not drive phosphoenzyme formation to completion. The maximal levels of phosphoenzyme that are formed at saturating concentrations of lumenal Ca2+ increase with increasing concentrations of Mg2+. This result requires that Ca2+ can bind to low-affinity lumenal sites on both the free enzyme and the phosphoenzyme, as well as to the high-affinity cytoplasmic calcium-binding sites. If there were no lumenal binding sites for Ca2+ on the free enzyme, high concentrations of lumenal Ca2+ would convert all of the enzyme to the same maximal concentration of Ca2.E approximately P.Mg at subsaturating concentrations of Mg2+ and Pi. We conclude that there are two low-affinity lumenal sites as well as two high-affinity cytoplasmic sites for Ca2+ on the free enzyme. Phosphorylation by ATP results in translocation of Ca2+ from the high-affinity to the low-affinity sites.

Nine-year-old Korean children (N = 299) were tested for reaction time (RT) and intelligence measured by Raven's Standard Progressive Matrices. Reaction times were broken into decision times and movement times and into three degrees of complexity. The results showed low but generally statistically significant correlations between decision times and intelligence. Generally, no significant differences existed between movement times and intelligence. Boys showed significantly faster movement times than girls did.