Collection of Czechoslovak Chemical Communications - digital archive 

Abstract

In the present study, we have investigated the possibility that aminophospholipid glycation-mediated perturbations of the POPE/POPC lipid bilayer structure affect the activity and structure of the membrane-bound Na⁺,K⁺-ATPase. It was found that both glucose and glyceraldehyde (GCA) reacted with aminophospholipid POPE, forming lipid-linked glycofluorophores with the absorbance and fluorescence properties of protein-linked AGEs. The lipid glycation was accompanied by progressive oxidative modification of unsaturated fatty acid residues. Measurements of the steady-state fluorescence anisotropy of TMA-DPH have been a first attempt at detecting distinctive bilayer structural perturbations induced by aminophospholipid glycation. The steady-state fluorescence anisotropy of TMA-DPH increased with the time of glycation, presumably because of the increased lipid order of the bilayer. To improve the definition of structural alterations of the glycated lipid bilayer, we attempted to measure the dynamics of TMA-DPH and DPH fluorescence. The effect of glycation was both to change the membrane dielectric constant (as probed by TMA-DPH and DPH fluorescence lifetimes) and increase the lipid order (as probed by time-resolved fluorescence anisotropy measurements). The aminophospholipid glycation reduced the activity of Na⁺,K⁺-ATPase, which was incorporated into glycated POPE/POPC vesicles. The enzyme inhibition correlated with the increase in the steady-state fluorescence anisotropy of TMA-DPH but not with the concentration of MDA (e.g., lipid oxidation). Therefore the inhibition of Na⁺,K⁺-ATPase activity induced by aminophospholipid glycation seems to be related to the modification of the protein molecule conformation through the lipid bilayer structure alterations. The inhibition of Na⁺,K⁺-ATPase activity was the sum of at least two factors: the increased lipid order and changed membrane dielectric constant. These factors can alter the lipid-lipid and lipid-protein interactions (e.g., electric multipole-multipole interactions) in membrane and thus provoke the inhibition of membrane bound enzymes.

Keywords: Fluorescence anisotropy; Membrane fluidity; ATPases; Lipid glycation; Lipid oxidation.