Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP, K+, Na+, and strophanthidin in vitro.
The effect of lipid peroxidation on the affinity of specific active sites of Na+,K+-ATPase for ATP (substrate), K+ and Na+ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100 microM ascorbate and 25 microM FeCl2 at 37 degrees C for time intervals from 0-20 min. The level of thiobarbituric acid reactive substances and the activity of Na+, K+-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0 +/- 3.8 to 8.8 +/- 0.9 mumol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2 +/- 0.1 to 16.5 +/- 1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K+ and Na+ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na+, K+-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K+ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na+, K+-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.
Mishra
O P
OP
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Delivoria-Papadopoulos
M
M
Cahillane
G
G
Wagerle
L C
LC
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D015227:Lipid Peroxidation / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013327:Strophanthidin / Q000378:metabolism*; D013552:Swine; D013858:Thiobarbiturates
D013858:Thiobarbiturates; D013327:Strophanthidin; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C029684:thiobarbituric acid; D011188:Potassium