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Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum.
- Source :
-
Biochimica et biophysica acta [Biochim Biophys Acta] 1989 Sep 18; Vol. 984 (3), pp. 326-38. - Publication Year :
- 1989
-
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 C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) 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 C12 E10 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.
- Subjects :
- Animals
Chromatography, High Pressure Liquid
Cross-Linking Reagents
Detergents
Fluorescein-5-isothiocyanate
Fluoresceins
Fluorescence Polarization
Glutaral
Membrane Proteins analysis
Molecular Weight
Phosphoric Monoester Hydrolases metabolism
Rabbits
Solubility
Thiocyanates
Calcium pharmacology
Calcium-Transporting ATPases metabolism
Sarcoplasmic Reticulum enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0006-3002
- Volume :
- 984
- Issue :
- 3
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta
- Publication Type :
- Academic Journal
- Accession number :
- 2550078
- Full Text :
- https://doi.org/10.1016/0005-2736(89)90300-3