Protein and lipid rotational dynamics in cardiac and skeletal sarcoplasmic reticulum detected by EPR and phosphorescence anisotropy.
We have used time-resolved phosphorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy to detect the rotational dynamics of the Ca-ATPase and its associated lipids in dog cardiac sarcoplasmic reticulum (DCSR), in comparison with rabbit skeletal SR (RSSR), in order to obtain insight into the physical bases for different activities and regulation in the two systems. Protein rotational motions were studied with time-resolved phosphorescence anisotropy (TPA) of erythrosin isothiocyanate (ERITC) and saturation-transfer EPR (ST-EPR) of a maleimide spin-label (MSL). Both labels were attached selectively and rigidly to the Ca-ATPase. Lipid rotational motions were studied with conventional EPR of stearic acid spin-labels. As in previous studies on RSSR, the phosphorescence anisotropy decays of both preparations at 4 degrees C were multiexponential, due to the presence of different oligomeric species. The rotational correlation times for the different rotating species were similar for the two preparations, but the total decay amplitude was substantially less for cardiac SR, indicating that more of the Ca-ATPase molecules are in large aggregates in DCSR. ST-EPR spectra confirmed that the Ca-ATPase is less rotationally mobile in DCSR than in RSSR. Lipid probe mobility and fatty acid composition were very similar in the two preparations, indicating that the large differences observed in protein mobility are not due to differences in lipid fluidity. We conclude that the higher restriction in protein mobility observed by both ST-EPR and TPA is due to more extensive protein-protein interactions in DCSR than in RSSR.
Birmachu
W
W
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Voss
J C
JC
Louis
C F
CF
Thomas
D D
DD
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D004285:Dogs; D004578:Electron Spin Resonance Spectroscopy; D004923:Erythrosine / Q000031:analogs & derivatives; D005454:Fluorescence Polarization; D017879:Isothiocyanates; D008055:Lipids / Q000737:chemistry*; D008301:Maleimides; D009124:Muscle Proteins / Q000737:chemistry*; D009132:Muscles / Q000737:chemistry*; D009206:Myocardium / Q000737:chemistry*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000737:chemistry*
D017879:Isothiocyanates; D008055:Lipids; D008301:Maleimides; D009124:Muscle Proteins; C043592:maleimide; C022232:erythrosine isothiocyanate; D000252:Calcium-Transporting ATPases; D004923:Erythrosine