FTIR study of ATP-induced changes in Na+/K+-ATPase from duck supraorbital glands.
The Na+/K+-ATPase uses energy from the hydrolysis of ATP to pump Na+ ions out of and K+ ions into the cell. ATP-induced conformational changes in the protein have been examined in the Na+/K+-ATPase isolated from duck supraorbital salt glands using Fourier transform infrared spectroscopy. Both standard transmission and attenuated total internal reflection sample geometries have been employed. Under transmission conditions, enzyme at 75 mg/ml was incubated with dimethoxybenzoin-caged ATP. ATP was released by flashing with a UV laser pulse at 355 nm, which resulted in a large change in the amide I band. The absorbance at 1659 cm(-1) decreased with a concomitant increase in the absorbance at 1620 cm(-1). These changes are consistent with a partial conversion of protein secondary structure from alpha-helix to beta-sheet. The changes were approximately 8% of the total absorbance, much larger than those seen with other P-type ATPases. Using attenuated total internal reflection Fourier transform infrared spectroscopy, the decrease in absorbance at approximately 1650 cm(-1) was titrated with ATP, and the titration midpoint K0.5 was determined under different ionic conditions. In the presence of metal ions (Na+, Na+ and K+, or Mg2+), K0.5 was on the order of a few microM. In the absence of these ions, K0.5 was an order of magnitude lower (0.1 microM), indicating a higher apparent affinity. This effect suggests that the equilibrium for the ATP-induced conformational changes is dependent on the presence of metal ions.
Pratap
Promod R
PR
Department of Physics and Astronomy, University of North Carolina at Greensboro, Greensboro, North Carolina 27402-6710, USA. pratapp@uncg.edu
Dediu
Oana
O
Nienhaus
G Ulrich
GU
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002448:Cell Adhesion; D004372:Ducks; D006868:Hydrolysis; D007477:Ions; D008051:Lipid Bilayers / Q000737:chemistry; D008956:Models, Chemical; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D012491:Salt Gland / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013997:Time Factors; D014466:Ultraviolet Rays
D007477:Ions; D008051:Lipid Bilayers; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium