Stopped flow and rapid quenching measurement of the transient steps induced by calcium binding to sarcoplasmic reticulum adenosine triphosphatase. Competition with Ca2+-independent phosphorylation.

Abstract

The transient steps induced by calcium binding to sarcoplasmic reticulum vesicles were concomitantly studied by stopped flow fluorometry and chemical quenching methods.The rate constant (hobs) for the transition between the forms of ATPase with low and high intrinsic fluorescence ("on" rate) was found to be 1.3 -t 0.4 s-' at pCa 4-2, pH 6, 20 "C, in the absence of KCI.A complete Robs uersus pCa curve was also obtained; magnesium concentrations from 0 to 20 m M had little effect on these hobs values.We designed a separate experimental setup allowing the study by chemical quenching methods of the transition between the two unphosphorylated forms of ATPase capable of reacting with Pi and ATP, respectively, In the absence of any possible activation by nucleotides, the observed rate constant for this transition was found to be 1.5 s-', a value consistent with the one derived from fluorometry.Addition of calcium to enzyme phosphorylated with Pi reduced the rate constant for sarcoplasmic reticulum ATPase transformation and subsequent phosphorylation by ATP (kobs 0.8 s-' at 5 n m M$+ and 20 n m phosphate).Under these conditions, the rate constant for fluorescence changes was also reduced.When the phosphoenzyme level rose under the initial conditions, the observed rate constant fell.In addition, detailed examination of the traces recorded indicated the presence of a second faster exponential of small amplitude.Different initial experimental conditions, in which calcium and phosphate were added simultaneously to the Ca2'-deprived enzyme, made this biphasic behavior more clearly evident.All these results are discussed in terms of the calcium binding mechanism, the acceleration by ATP of the Ca2+ binding-induced transition, and the competition between the calcium-induced transition and the calcium-independent phosphorylation by Pi.Calcium is transported across sarcoplasmic reticulum membranes by a calcium-dependent ATPase.'The hydrolytic cycle +