Calcium fluxes in sarcoplasmic reticulum

In an effort to understand the processes of calcium uptake and release in sarcoplasmic reticulum (SR) of skeletal muscle, studies were performed on the purified (Ca2+ -Mg2+ )ATPase of SR, native SR membrane and a reconstituted system of the (Ca2+ -Mg2+ )ATPase in phospholipid vesicles. The effect of pH, calcium concentration and the non-ionic detergent C12E8 on the ATPase activity of the (Ca2+ -Mg2+ )ATPase were examined, and the results explained on the basis of a detailed kinetic model of the enzyme. A procedure for the reconstitution of the (Ca2+ -Mg2+ )ATPase into sealed phospholipid vesicles was developed in order to study the effects of calcium uptake. Using this system it was shown that the ATPase mediates a rapid efflux of calcium from the vesicles, that is dependent on external calcium concentration, pH, external magnesium concentration and adenine nucleotides, and a kinetic scheme for this efflux is proposed; the effects of phsopholipid structure on the process of calcium uptake have been explained on the basis of changes in the rate of this efflux. It was further shown that the ATPase is not the site of the K+ and Pi channels known to exist in native SR, and that myo inositol triphosphate does not influence the rate of this calcium efflux. The process of calcium uptake and release from native SR vesicles was examined, and the release was found to occur in a biphasic fashion. It was shown that the onset of the second release phase coincides with the decline in ATP levels in the bathing media to levels approaching the Km of the ATPase for ATP, and that the first release phase has characteristics similar to those of the rapid calcium efflux previously described. The effect of pH, temperature and maleic acid on calcium uptake and release have been rationalised. The ability of AgNO3 to induce calcium release has been shown to be the result of Ag+ interacting with the ATPase. The effect of diet on the fatty acyl chain composition of SR phospholipid was examined, and the effects of such changes on in vitro SR function shown to be limited.