Abstract
Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.
Highlights
The platelet protein resulted in the appeaorfanpcreod- Theseandother differences in the plateletand skeletal ucts with apparent molecular masses of 70 kDa, 39 muscle systems suggest that a better understandinogf calcium kDa, and 31 kDa, while asimilardigestion ofthe movements in these cells can be obtained by comparative sarcoplasmic reticulum calciumpump protein yielded studies
54-kDa, 52.5-kDa, 46-kDa, 41-kDanad, 36-kDa frag- tional properties of the platelet microsome and skeletal musments.Exposureofthesarcoplasmicreticulumand plateletCa2+-ATPaseproteinsto S. aureus protease yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPasesfrom platelets cle sarcoplasmic reticulum Ca2+-ATPases.The results demonstrate important structural and functional differences between the two proteins
The platelet Ca2+-ATPase and itsproteolytic products were detected with the immunodetection procedure, while the sarcoplasmic reticulum calcium pump protein and relatedproteolytic fragments were detected with Coomassie Brilliant Blue stain ofgels
Summary
Eukaryotic cells have evolved precise mechanisms for regulating intracellularlevels of calcium. These mechanisms help cells maintain high gradients of calcium across their plasma membranes with calcium concentrations inside the cell several thousandfold less than calcium concentrations in the extracellular fluid. They provide methods, especially in excitable cells, whereby levels of intracellular calcium can be increased rapidly but ingraded fashion. This typically involves release of calcium from various subcellular stores so that thecell is not exposed to thehigh levels of extracellular calcium. The mechanisms involved in these processes are unique from cell type to cell type and, to a certain extent, reflect the specialized function of the cell
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