Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.
In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.
Vasić
Vesna
V
Department of Physical Chemistry, Vinca Institute of Nuclear Sciences, 11001 Belgrade, Yugoslavia. evasic@rt270.vin.bg.ac.yu
Jovanović
Danijela
D
Horvat
Anica
A
Momić
Tatjana
T
Nikezić
Gordana
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002104:Cadmium / Q000494:pharmacology*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology*; D011137:Polystyrenes / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism; D013997:Time Factors
D004800:Enzymes, Immobilized; D011137:Polystyrenes; D002104:Cadmium; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D008628:Mercury