FTIR Study of ATP-Induced Changes in Na+/K+-ATPase from Duck Supraorbital Glands

Abstract

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.

Modulation of Reconstituted Pig Kidney Na+/K+-ATPase Activity by Cholesterol in Endogenous Lipid Vesicles: Role of Lipid Domains

“…In this regard, Chetverin et al indicated that according to their results the enzyme working cycle lacks gross structure changes (85). On the other hand, Pratap et al, reporting an investigation related to ATP-induced conformational changes in Na + /K + -ATPase, indicated that from their FTIR results it is not possible to decide if these changes correspond to large changes localized in the cytoplasmic loop between the M4 and M5 transmembrane domains or a sum of smaller changes that occurred throughout the protein (86).…”

Spectroscopic investigation of the reaction mechanism of CopB‐B, the catalytic fragment from an archaeal thermophilic ATP‐driven heavy metal transporter

“…Ca-ATPase and Na ⁄ K-ATPase have been extensively investigated by time-resolved FTIR absorbance difference spectroscopy using various nucleotides and nucleotide analogues [7][8][9][10][11][12][13]. These studies have suffered from the fact that the described mammalian proteins could only be purified from native tissue material.…”

Change of Erythrocytes Transport Function in Blood of Men with Prostate Adenocarcinoma (before and after Plastic Orchiectomy)

“…It is established that there is a two-side relationship between Na + /K + -ATP-ase activity and concentration of Na + and K + ions. In particular, Na + /K + -ATP-ase is the main enzyme regulating the concentration gradient of Na + and K + ions in intra-and extra-cellular areas; But mentioned ions, in turn, play the role of enzyme's regulators [1] [16].…”

“…Na + \K + -ATP-ase belongs to the transporter family of P-type ATP-ases [1]. It's known that the enzyme performs the signaling function in addition to the transport function [1] [2].…”

“…Na + \K + -ATP-ase belongs to the transporter family of P-type ATP-ases [1]. It's known that the enzyme performs the signaling function in addition to the transport function [1] [2]. Na + /K + -ATP-ase interacts with neighboring membrane proteins and organized cytosolic cascades of signaling proteins to send messages to the intracellular organelles [3] and thus effects the expression of various genes, proliferation of cells, activation of some hormones and growth factors and phosphorylation of certain proteins [4].…”