INFLUENCE OF POLY(ETHYLENE GLYCOL) AND AQUEOUS VISCOSITY ON THE ROTATIONAL DIFFUSION OF MEMBRANOUS NA,K-ATPASE

The Na,K-ATPase [ATP phosphohydrolase (Na+/K+-transporting), E.C. 3.6.1.37] in native membranes from the salt gland of Squalus acanthias has been spin-labeled covalently with a chloromercuri nitroxide derivative, and the rotational diffusion of the protein has been studied, as a function of the concentration of glycerol or poly(ethylene glycol) in the suspending medium, by means of saturation-transfer electron spin resonance spectroscopy. The effective rotational correlation time of the protein increases linearly with the viscosity of the aqueous glycerol medium, with a gradient whose value indicates that ca. 50-70% of the volume of the Na,K-ATPase protein is external to the membrane. The effective rotational correlation times of the protein in poly(ethylene glycol) solutions are considerably greater than those in glycerol solutions of the same viscosity and increase nonlinearly with the viscosity of the suspending medium, indicating that increasing concentrations of poly(ethylene glycol) induce aggregation of the integral proteins within the membrane. The value reached at 50% poly(ethylene glycol) corresponds to a degree of aggregation of the proteins between 2 and 5 depending on whether the ethylene glycol polymer is excluded from the membrane surface region. The results are discussed with respect to hydration forces and poly(ethylene glycol)-induced cell fusion.