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Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle
D A Ferrington1, T E Jones, Z Qin
1Department of Biochemistry, University of Kansas, Lawrence 66045-2106, USA.
Abstract:
Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.
Insights
Aging causes sarcoplasmic reticulum Ca-ATPase in rat muscle to become less stable and prone to self-association when heated, despite no initial changes in activity or expression.
Area of Science:
- Biochemistry
- Molecular Biology
- Gerontology
Background:
- Sarcoplasmic reticulum Ca-ATPase (SERCA) is crucial for muscle function.
- Aging may affect SERCA stability and structure.
- Understanding age-related changes in SERCA is vital for muscle health.
Purpose of the Study:
- To investigate age-related differences in sarcoplasmic reticulum Ca-ATPase from young and aged rats.
- To compare functional and structural properties, including heat stability and self-association.
- To identify molecular mechanisms underlying age-associated functional decline.
Main Methods:
- Purification of sarcoplasmic reticulum membranes from young and aged Fischer 344 male rats.
- Measurement of Ca-ATPase activity, calcium transport, and protein expression levels.
- Saturation-transfer EPR spectroscopy for Ca-ATPase self-association and lipid dynamics.
- Assessment of heat inactivation and tryptic digestion rates.
Main Results:
- No age-related differences in Ca-ATPase expression, calcium transport, or ATPase activity were observed.
- Ca-ATPase from aged muscle showed more rapid heat inactivation and self-association into inactive oligomers.
- Increased solvent exposure of the Ca-ATPase cytoplasmic domain in aged muscle was detected via tryptic digestion.
- Heat inactivation amplified structural differences, with faster unfolding in aged Ca-ATPase.
Conclusions:
- Age-related alterations in Ca-ATPase structure, specifically increased solvent exposure, contribute to reduced conformational and functional stability.
- These structural changes in aged muscle Ca-ATPase lead to increased susceptibility to heat-induced inactivation and self-association.
- Lipid bilayer dynamics and protein-lipid interface interactions were not significantly affected by age or heat inactivation.