Isolation and characterization of a stable Chinese hamster ovary cell line overexpressing the plasma membrane Ca(2+)-ATPase.
Stable Chinese hamster ovary (CHO) cell lines overexpressing the human plasma membrane Ca(2+)-ATPase (PMCA) were generated, and three independent cell clones were characterized in details. They overexpressed high amounts of active PMCA pump (15-20 times over the amount of endogenous PMCA) as indicated by experiments in which the formation of the phosphoenzyme intermediate and the uptake of Ca2+ by microsomes were measured. Immunocytochemistry experiments coupled to the biotinylation of the pump in the intact cells indicated the correct deliver of the expressed pump to the plasma membrane. The expressed pump was purified by affinity chromatography on calmodulin sepharose. The PMCA of transfected CHO cells promoted an increase of Ca2+ into the medium, after induction of Ca2+ release from the internal stores by activation of a purinergic receptor. An evident decrease of the activity of the endogenous sarcoplasmic reticulum Ca(2+)-ATPase pump was observed, probably related to the down-regulation of its expression. The cells overexpressing the PMCA pump had delayed recovery after trypsinization and plating. Their doubling time was, however, the same as CHO cells.
Guerini
D
D
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.
Schröder
S
S
Foletti
D
D
Carafoli
E
E
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014162:Transfection
D000252:Calcium-Transporting ATPases; D002118:Calcium