Difference between the E1 and E2 conformations of gastric H+/K+-ATPase in a multilamellar lipid film system - Characterization by fluorescence and ATR-FTIR spectroscopy under a continuous buffer flow

A liquid flow cell was used for an attenuated total reflection-Fourier tran sform infrared spectroscopy (ATR-FTIR) study of conformational changes taki ng place in the gastric H+/K+-ATPase. Shifting from E1 to E2 form is induce d by replacing Na+ by K+ ions. Introducing ions through a flow passing over a protein multilayer film induced the conformational change without cell m anipulations. Measurement sensitivity was thereby improved by about one ord er of magnitude. The detection threshold allowed the possibility to detect a change affecting five amino acids out of the 1324 that compose the H+/K+- ATPase molecule. It appeared that fewer than five amino-acid residues under go a conformational change upon replacing Na+ by K+ ions in the medium. Evi dence that conformational changes occur in an identical system was brought by monitoring the fluorescence of fluorescein isothiocyanate-labeled H+/K+- ATPase in similar conditions. Our data suggest that essentially the tertiar y structure of the protein is modified.