Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.
Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.
Esmann
M
M
Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Federal Republic of Germany.
Marsh
D
D
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D003902:Detergents; D004058:Diffusion; D004284:Dogfish; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D007700:Kinetics; D012491:Salt Gland / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
D003902:Detergents; D000254:Sodium-Potassium-Exchanging ATPase