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 transform infrared spectroscopy (ATR-FTIR) study of conformational changes taking place in the gastric H+/K+-ATPase. Shifting from E1 to E2 form is induced by replacing Na+ by K+ ions. Introducing ions through a flow passing over a protein multilayer film induced the conformational change without cell manipulations. Measurement sensitivity was thereby improved by about one order 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 undergo a conformational change upon replacing Na+ by K+ ions in the medium. Evidence 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 tertiary structure of the protein is modified.
Vander Stricht
D V
DV
Laboratoire de Chimie-Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Raussens
V
V
Oberg
K A
KA
Ruysschaert
J M
JM
Goormaghtigh
E
E
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D005748:Gastric Fundus / Q000737:chemistry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D007477:Ions; D008055:Lipids / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012964:Sodium / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013552:Swine
D007477:Ions; D008055:Lipids; D012964:Sodium; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium