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

R. Heim, T. Iwata, E. Zvaritch, H. P. Adamo, B. Rutishauser, E. E. Strehler, D. Guerini, E. Carafoli

Research output: Contribution to journalArticle

56 Scopus citations

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 so-called 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 K(M) (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 - Jan 1 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Heim, R., Iwata, T., Zvaritch, E., Adamo, H. P., Rutishauser, B., Strehler, E. E., 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, 267(34), 24476-24484.