ATP synthesis by sarcoplasmic reticulum ATPase following Ca2+, PH, temperature, and water activity jumps.

Phosphorylation of sarcoplasmic reticulum ATPase with Pi and phosphoryl transfer from the phosphoenzyme to ADP to form ATP were studied in experiments including two sequential steps in the absence of a transmembrane Ca2+ gradient. This was accomplished with a variety of experimental manipulations including jumps of Ca2+ concentrations, pH, temperature, and water activity. Phosphorylation with Pi requires dissociation of Ca2+ from “high affinity’’ sites of the enzyme and it is favored by low pH (6.0), high temperature (37 “C), and a reduced water activity. The latter condition is achieved by addition of miscible organic solvents in high concentrations (e.g. dimethyl sulfoxide, 4076, v/v) to the medium. Phosphoryl transfer from phosphoenzyme to ADP requires addition of Ca2+ in concentrations that are dependent on pH and temperature and occurs optimally in totally aqueous media (high water activity). Intermediate dimethyl sulfoxide concentrations (20%, v/v) allow both the phosphorylation and the phosphoryl transfer reaction to occur, and the latter can be simply started by addition of ADP and Ca2+ to the enzyme incubated with Pi in the absence of Ca”. ATP formation through phosphoryl transfer from phosphoenzyme to ADP requires addition of Ca2+ concentrations sufficiently high to saturate “low affinity” sites in the phosphoenzyme. However, at alkaline pH (8.0) and low temperature (0 “C), ATP synthesis can be obtained even in the presence of low Ca2+ concentrations, in a range which is only sufficient to saturate high affinity sites. The commonly observed requirement for calcium occupancy of low affinity sites is interpreted as a step permitting equilibration between a phosphoenzyme with low phosphorylation potential and binding sites in a low affinity state and a phosphoenzyme with high phosphorylation potential and binding sites in a high affinity state. It is then postulated that high and low affinity binding sites are expressions of two interconverting states of the phosphoenzyme. At alkaline pH and low temperature, proton dissociation from enzyme residues permits transformation of the sites from the