Fourier transform infrared spectroscopic studies on gastric H+/K+-ATPase.
Suspensions of membrane-bound H+/K+-ATPase in both H2O and 2H2O were investigated using Fourier transform infrared (FT-IR) spectroscopy. Second-derivative techniques were used to reveal the overlapping bands in the 1800-1500 cm-1 region. Analysis of the amide I band shows that the protein component contains substantial amounts of both alpha-helical and beta-sheet structures. Addition of 10 mM KCl to a suspension in 2H2O does not significantly affect the amide I band, indicating that the E1-E2 conformational transition of the enzyme, induced by K+, does not involve a gross change in protein secondary structure. Analysis of the amide II band in the spectra of suspensions in 2H2O shows that inhibition of the enzyme with omeprazole increases the rate of 1H-2H exchange, indicating an increase in conformational flexibility. Furthermore, an additional feature at 1628 cm-1 in the spectra of the inhibited samples in 2H2O could either support a conformational change or arise from a vibrational mode of omeprazole in its enzyme-bound form. The frequency of the band due to the symmetric stretching vibrations of the methylene groups of the lipid acyl chains increases steadily with increasing temperature indicating that there is no co-operative melting process in the lipid component of the membrane over the temperature range 9-50 degrees C. For comparison, FT-IR studies on aqueous suspensions of Na+/K+-ATPase were also carried out. These show that the protein components in the Na+/K+- and H+/K+-ATPases have similar secondary structures.
Mitchell
R C
RC
Smith Kline & French Research Limited, Welwyn, Hertfordshire, U.K.
Haris
P I
PI
Fallowfield
C
C
Keeling
D J
DJ
Chapman
D
D
D000251:Adenosine Triphosphatases; D000818:Animals; D005583:Fourier Analysis; D017506:H(+)-K(+)-Exchanging ATPase; D006860:Hydrogen Bonding; D008565:Membrane Proteins; D009853:Omeprazole / Q000494:pharmacology; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D013270:Stomach / Q000201:enzymology*; D013552:Swine; D013696:Temperature; D014867:Water
D008565:Membrane Proteins; D014867:Water; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D009853:Omeprazole