Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment

Roger Heim, Tomoko Iwata, Elena Zvaritch, Hugo P. Adamo, Barbara Rutishauser, Emanuel E. Strehler, Danilo Guerini, Ernesto Carafoli

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

Isoform 4b of the human plasma membrane Ca2+ pump was expressed in COS cells and in the baculovirus system (Sf9 cells). A 105-kDa pump fragment lacking the first two transmembrane domains and the socalled transduction domain was also expressed. The expression level was 2-4 times the background in COS cells and at least 7 times in the baculovirus system. Tests on membranes from both systems showed that the expressed pump was active. The expressed pump and the 105-kDa fragment were isolated from Sf9 cell membranes by calmodulin affinity chromatography. The pump had Ca2+-dependent ATPase activity with a calmodulin stimulation factor of 3, formed a La3+-stabilized phosphoenzyme, and had a KM (Ca2+) in the presence of calmodulin of about 1 μM. The 105-kDa fragment, assayed by the phosphoenzyme test on COS or Sf9 cell membranes or by ATPase measurements after isolation from Sf9 cells, proved inactive. Laser confocal microscopy on Sf9 cells showed that both the pump and the 105-kDa fragment were apparently associated with the plasma membrane. The expressed pump in COS and Sf9 cells and the endogenous pump in a number of other cell lines had a slower gel mobility (i.e. a higher apparent molecular mass) than the erythrocyte pump.

Original languageEnglish (US)
Pages (from-to)24476-24484
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number34
StatePublished - Dec 5 1992
Externally publishedYes

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Sf9 Cells
Cell membranes
Purification
COS Cells
Cell Membrane
Pumps
Calmodulin
Baculoviridae
Confocal Microscopy
Adenosine Triphosphatases
Calcium-Transporting ATPases
Affinity Chromatography
Affinity chromatography
Protein Isoforms
Confocal microscopy
Erythrocytes
Gels
Molecular mass
Cell Line
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Heim, R., Iwata, T., Zvaritch, E., Adamo, H. P., Rutishauser, B., Strehler, E. E., ... Carafoli, E. (1992). Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. Journal of Biological Chemistry, 267(34), 24476-24484.

Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. / Heim, Roger; Iwata, Tomoko; Zvaritch, Elena; Adamo, Hugo P.; Rutishauser, Barbara; Strehler, Emanuel E.; Guerini, Danilo; Carafoli, Ernesto.

In: Journal of Biological Chemistry, Vol. 267, No. 34, 05.12.1992, p. 24476-24484.

Research output: Contribution to journalArticle

Heim, R, Iwata, T, Zvaritch, E, Adamo, HP, Rutishauser, B, Strehler, EE, Guerini, D & Carafoli, E 1992, 'Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment', Journal of Biological Chemistry, vol. 267, no. 34, pp. 24476-24484.
Heim R, Iwata T, Zvaritch E, Adamo HP, Rutishauser B, Strehler EE et al. Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. Journal of Biological Chemistry. 1992 Dec 5;267(34):24476-24484.
Heim, Roger ; Iwata, Tomoko ; Zvaritch, Elena ; Adamo, Hugo P. ; Rutishauser, Barbara ; Strehler, Emanuel E. ; Guerini, Danilo ; Carafoli, Ernesto. / Expression, purification, and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. In: Journal of Biological Chemistry. 1992 ; Vol. 267, No. 34. pp. 24476-24484.
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