AMINOPHOSPHOLIPID GLYCATION CAUSES LIPID BILAYER STRUCTURE ALTERATIONS AND INHIBITION OF MEMBRANE-BOUND NA-ATPASE(,K+)

In the present study, we have investigated the possibility that aminop hospholipid glycation-mediated perturbations of the POPE/POPC lipid bi layer structure affect the activity and structure of the membrane-boun d Na+,K+-ATPase. It was found that both glucose and glyceraldehyde (GC A) reacted with aminophospholipid POPE, forming lipid-linked glycoflu orophores with the absorbance and fluorescence properties of protein-l inked AGEs. The lipid glycation was accompanied by progressive oxidati ve modification of unsaturated fatty acid residues. Measurements of th e steady-state fluorescence anisotropy of TMA-DPH have been a first at tempt at detecting distinctive bilayer structural perturbations induce d by aminophospholipid glycation. The steady-state fluorescence anisot ropy of TMA-DPH increased with the time of glycation, presumably becau se of the increased lipid order of the bilayer. To improve the definit ion of structural alterations of the glycated lipid bilayer, we attemp ted to measure the dynamics of TMA-DPH and DPH fluorescence. The effec t of glycation was both to change the membrane dielectric constant (as probed by TMA-DPH and DPI-I fluorescence lifetimes) and increase the lipid order (as probed by time-resolved fluorescence anisotropy measur ements). The aminophospholipid glycation reduced the activity of Nai,K +-ATPase, which was incorporated into glycated POPE/POPC vesicles. The enzyme inhibition correlated with the increase in the steady-state fl uorescence anisotropy of TMA-DPH but not with the concentration of MDA (e.g., lipid oxidation). Therefore the inhibition of Na+,K+-ATPase ac tivity induced by aminophospholipid glycation seems to be related to t he modification of the protein molecule conformation through the lipid bilayer structure alterations. The inhibition of Na+,K+-ATPase activi ty was the sum of at least two factors: the increased lipid order and changed membrane dielectric constant, These factors can alter the lipi d-lipid and lipid-protein interactions (e.g., electric multipole-multi pole interactions) in membrane and thus provoke the inhibition of memb rane bound enzymes.