Monomeric and trimeric structures of active Na,K-ATPase in C12E8 solution

Horse kidney Na,K-ATPase solubilized with dodecyloctaethyleneglycolether was subjected to high performance gel chromatography (HPLC) on TSK G 4000 SW in the presence of 0.01% C12E8. Successive on-line measurements of low angle laser light scattering (LS) and refractive index (RI) were made, and values of refractive index increment (dn/dc) were measured with the same differential refractometer under the same conditions. Two peaks (peak-2 and peak-3) with Na,K-ATPase activity besides that at the void volume were detected in the HPLC effluent fractions, and from their (dn/dc) values and the linear plot of protein standards, the M.W.s of these peaks were calculated to be roughly 535K and 175K respectively. Both peaks showed alpha and beta, but not gamma, bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The activity peak fractions obtained after glycerol density gradient centrifugation to remove detergent micelles showed HPLC peak-2 and peak-3. The ratios of (Output) LS/(Output)RI of peak-2 to that of peak-3 and (Output) LS/(Output)UV 280 of the peak-2 to that of peak-3 were both nearly 3. The above findings and the results of electron microscopy of findings and the results of electron microscopy of negatively stained preparations strongly suggested that peak-2 and peak-3 of enzyme activity consist of the trimer (alpha beta)3 and the monomer (alpha beta), respectively.