Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosine triphosphatase in membranes from Squalus acanthias

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Abstract
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.degree. 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.degree. 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.degree. C, and the activity decreases with a half-life of ca. 70 min on incubation at 37.degree. 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. The effective rotational correlation time of the membranous enzyme, deduced from the low-field and high-field spectral line-height ratios using calibrations from isotropically rotating spin-labeled hemoglobin, lies in the region of 50 .mu.s, implying an upper limit of .tau.R.dblvert. = 25 .mu.s for the true rotational correlation time of the protein.