The long term objectives of the proposed research are to explain the role of calmodulin in the reaction mechanism of the red blood cell membrane Ca2+- ATPase and the mechanism by which calmodulin modulates the enzyme. To achieve the long term objectives, the following specific aims have been set for this project. Initially, the optimal conditions for studying the modulation of the enzyme action by calmodulin will be determined. This will be the first characterization of the influence of temperature, and enzyme:detergent ratio, on the calmodulin stimulation. The main part of the proposal will be characterization of the partial reactions of the catalytic mechanism of the purified Ca2+-ATPase, and the role of calmodulin in its regulation. From such studies it will be known for the first time which partial reactions of the ATPase are regulated by calmodulin. Structural studies will then be done to test the hypothesis that two ATPase forms are produced, a form with calmodulin stimulated activity and a form with calmodulin independent activity. These forms are postulated to be monomers and dimers, respectively. Possible calmodulin facilitated interaction of enzyme units will be investigated. The Ca2+-ATPase will be purified from the membrane by calmodulin affinity column chromatography in the presence of the nonionic detergent, dodecyl octaethylene glycol monoether (C12E8). The two ATPase forms will be obtained by changing the ATPase to detergent ratio in the assay mixture. For studies of the reaction cycle, rapid kinetic methods will be employed to resolve the partial reactions occurring in the millisecond time scale. Determination of oligomeric states will be done by combination of three techniques: polyacrylamide gel electrophoresis, gel filtration and chemical cross-linking.
