Purification of the microsomal Ca2(+)-ATPase from rat liver.
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.
Jong
Y J
YJ
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
Sheldon
A
A
Zhang
G H
GH
Kraus-Friedmann
N
N
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004795:Enzyme Stability; D000939:Epitopes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
D000939:Epitopes; D000252:Calcium-Transporting ATPases