Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosinetriphosphatase in membranes from Squalus acanthias.
The sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)-ATPase] in membranous preparations from Squalus acanthias has been spin-labeled on sulfhydryl groups after prelabeling with N-ethylmaleimide. Saturation-transfer electron spin resonance spectroscopy has been used to study the rotational motions of the labeled protein on the microsecond time scale. Effective rotational correlation times deduced from the diagnostic line-height ratios in the second-harmonic, 90 degrees out-of-phase (V2') spectra are much larger than those deduced from the spectral integrals, indicating the presence of large-scale segmental motions, in addition to rotation of the protein as a whole. Experiments involving controlled cross-linking of the protein by glutaraldehyde, as well as measurements of the line broadening of the conventional electron spin resonance spectra, support this interpretation. Both the spectral integrals and diagnostic line-height ratios are found to increase irreversibly with time on incubation at temperatures greater than 20 degrees C, corresponding to a decrease in the segmental motion of the protein and probably also in the overall protein rotation. The native enzyme displays a marked nonlinearity in the Arrhenius temperature dependence of the activity at temperatures above 20 degrees C, and the activity decreases with a half-life of ca. 70 min on incubation at 37 degrees C (but not on incubation at low temperature), paralleling the time- and temperature-dependent changes in the saturation-transfer spectra of the labeled protein. Both of these observations suggest that the changes observed in the molecular dynamics could correspond to functional properties of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Esmann
M
M
Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Federal Republic of Germany.
Horváth
L I
LI
Marsh
D
D
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004284:Dogfish; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D012007:Rectum / Q000201:enzymology; D012627:Sebaceous Glands / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013816:Thermodynamics
D000254:Sodium-Potassium-Exchanging ATPase