Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

Tamas Keresztes, Istvan Jona, Slawomir Pikula, Miklos Vegh, Nandor Mullner, Sandor Papp, Anthony Martonosi

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Abstract

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5′-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12E8 or Brij 36T (≊ 4 mg/mg prottein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.

Original languageEnglish
Pages (from-to)326-338
Number of pages13
JournalBBA - Biomembranes
Volume984
Issue number3
DOIs
Publication statusPublished - Sep 18 1989

Fingerprint

Calcium-Transporting ATPases
Sarcoplasmic Reticulum
Oligomers
Calcium
Adenosine Triphosphatases
Molecules
Detergents
Fluorescein-5-isothiocyanate
Fluorescence Polarization
Fluorescence
Dimers
Crosslinking
High performance liquid chromatography
Glutaral
Polarization
Membranes
High Pressure Liquid Chromatography
Biochemistry
Energy Transfer
Energy transfer

Keywords

  • ATPase dimer
  • ATPase, Ca-
  • Calcium ion effect
  • Sarcoplasmic reticulum

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology

Cite this

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum. / Keresztes, Tamas; Jona, Istvan; Pikula, Slawomir; Vegh, Miklos; Mullner, Nandor; Papp, Sandor; Martonosi, Anthony.

In: BBA - Biomembranes, Vol. 984, No. 3, 18.09.1989, p. 326-338.

Research output: Contribution to journalArticle

Keresztes, T, Jona, I, Pikula, S, Vegh, M, Mullner, N, Papp, S & Martonosi, A 1989, 'Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum', BBA - Biomembranes, vol. 984, no. 3, pp. 326-338. https://doi.org/10.1016/0005-2736(89)90300-3
Keresztes, Tamas ; Jona, Istvan ; Pikula, Slawomir ; Vegh, Miklos ; Mullner, Nandor ; Papp, Sandor ; Martonosi, Anthony. / Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum. In: BBA - Biomembranes. 1989 ; Vol. 984, No. 3. pp. 326-338.
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