Abstract
The Ca2(+)-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficoll-sucrose treatment, column chromatography with agarose-hexane adenosine 5'-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca2(+)-ATPase was stable for at least two weeks when stored at -70 degrees C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca2(+)-ATPase. Further characterization of the ER Ca2(+)-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca2(+)-ATPase cross-reacted with the purified Ca2(+)-ATPase from rat liver ER membranes.
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