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°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.
Bibliographical noteFunding Information:
We thank Dr. Bruce Cutler for his help in obtaining the electron micrographs and Dr. Jack Schlager for advice on statistical analysis. This work was supported by the Scientific Education Partnership, a Marion Merrell Dow Foundation, the American Federation for Aging Research, and the National Institute of Aging (grant no. AG12275).
- Calcium regulation
- Fischer 344 rat
- Sarcoplasmic reticulum Ca-ATPase
- Skeletal muscle
- Spin label