Collect. Czech. Chem. Commun. 1996, 61, 160-170

Effect of Enzymically Generated Electronic-Excited Species on Membrane-Bound Na+,K+-ATPase

Tomáš Obšila, Evžen Amlerb and Zdeněk Pavlíčeka

a Department of Physical and Macromolecular Chemistry, Charles University, 128 40 Prague 2, Czech Republic
b Institute of Physiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic


The effect of electronic-excited species generated by systems 2-methylpropanal (MPAL) plus horse-radish peroxidase (HRP) and glyceraldehyde (GCA) plus HRP on Na+,K+-ATPase activity was investigated. For the system MPAL-HRP (which generates triplet acetone) Na+,K+-ATPase activity, the concentration of malondialdehyde, the membrane lateral pressure and the collisional quenching of protein tryptophan residues by NaI were determined. The enzyme activity decreased by 70% during the incubation. Lipid peroxidation products were increased to a steady state level from 5.5 ± 0.5 to 22.6 ± 0.7 nmol malondialdehyde mg-1 protein after 60 min. The membrane lateral pressure was increased from 83.6 ± 0.4 to 90 ± 0.9 mN m-1. In peroxidized membranes, the fluorescence intensity of tryptophan residues was decreased and the fluorescence intensity of bityrosine adducts was increased. Changes of conformation of Na+,K+-ATPase were observed by the collisional quenching of protein tryptophan residues by NaI. For the system GCA-HRP the Na+,K+-ATPase activity decreased by 20% after incubation with GCA and by 30% with GCA-HRP. Changes of conformation of Na+,K+-ATPase were detected both after the incubation with GCA and GCA-HRP. These results demonstrate that electronic-excited species inhibit the activity of membrane-bound Na+,K+-ATPase both by an increase of membrane lateral pressure of the lipid microenvironment of the ATPase molecules and by a direct effect of oxidation of protein residues, i.e. tryptophan, tyrosine.

Keywords: Electronic-excited species; Na+,K+-ATPase; Fluorescence.