We have undertaken a detailed examination of changes associated with aging in lipid composition and corresponding physical properties of hindlimb skeletal sarcoplasmic reticulum (SR) membranes isolated from young (5 months), middle-aged (16 months), and old (28 months) Fischer strain 344 rats. Silica gel HPLC chromatography was used to separate phospholipid headgroup species. Subsequent reversed-phase HPLC was used to resolve fatty acid chain compositions of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species. For all three phospholipid pools, significant age-related variations are observed in the abundance of multiple molecular species, particularly those having polyunsaturated fatty acid chains. Using mass spectrometry (fast atom bombardment and tandem techniques) to distinguish ester- from ether-linked phosphatidylethanolamine species, we demonstrate that overall plasmenylethanolamine content is substantially increased with age, from 48 mol% to 62 mol%. A substantial increase is also observed in the single molecular species 18:0-20:4 phosphatidylinositol suggesting implications for signalling pathways. In addition, associated with senescence we find a significant increase in the rigidifying lipid, cholesterol. Despite these changes in lipid composition of different aged animals, the average bilayer fluidity examined at several bilayer depths with stearic acid spin labels, is not altered. Neither do we find differences in the rotational mobility of maleimide spin-labeled Ca2+-ATPase, as determined from saturation-transfer electron paramagnetic resonance, which is sensitive to both the fluidity of lipids directly associated with the Ca2+-ATPase and to its association with proteins.
Bibliographical noteFunding Information:
We wish to thank Mr. Robert Drake for acquisition of FAB spectra. The tandem mass spectrometer was purchased with the aid of a National Institutes of Health grant S10 RRO 6294-01 and the University of Kansas. This work was supported by grants from the Scientific Education Partnership, a Marion Merrell Dow Foundation and NIH GM46837. A.G.K. is a Postdoctoral Fellow of Marion Merrell Dow Foundation; on leave from the Biophysical Group, Institute of Chemical Kinetics and Combustion, Novosibirsk, 630090, Russia.
- ATPase, Ca-
- Mass spectrometry
- Membrane composition
- Sarcoplasmic reticulum
- collisionally-induced dissociation
- electron paramagnetic resonance
- fast atom bombardment
- high-performance liquid chromatography
- magic bullet (matrix for mass spectrometry)
- phosphatidic acid
- sarcoplasmic reticulum
- saturation transfer electron paramagnetic resonance
- stearic acid spin label