Effect of pH and different substrates on the electrokinetic properties of (Na+, K+)-ATPase vesicles.
Some biophysical properties of a (Na+, K+)-ATPase preparation from guinea-pig kidney have been analysed. The recently developed technique of laser Doppler spectroscopy was applied to measure particle mobility under electrophoretic conditions. The following results were obtained: 1. magnesium ions at pH 7.3 decrease the mobility of the ATPase containing vesicles by binding to negatively charged surface groups. At pH 3.3 the competitive binding of protons causes a shift of the mobility vs. [Mg2+] curve to higher values of [Mg2+], 2. binding of ATP at pH 7.3 (Kd = 0.9 X 10(-4) M for (mM 1 NaCl, 0.2 KCl, 0.1 MgCl2, 0.1 Tris) was measured as an increase in particle mobility depending also on [Mg2+]. At pH 3.3 also unspecific ATP-binding occurred, 3. ITP and GTP had the same Kd value as ATP; ADP a slightly lower one (Kd = 1.2 X 10(-4) M). Tris-H3PO4 (Kd = 2.6 X 10(-4) M) was also able to increase particle mobility, but only at higher concentrations and not to the same extent as ATP; AMP induced only very small changes, 4. from the mobility-pH curve an isoelectric point of 4.1 is derived (buffer: 1 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl2, 0.1 mM Tris). In the presence of 0.9 mM ATP the isoelectric point is shifted to 3.2. As the electrophoretic mobility is directly proportional to the net charge of the vesicles, the results may be interpreted as changes in surface charge density, originating from both a conformational change of the ATPase polypeptide and a decrease in vesicle size.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004573:Electrolytes; D004586:Electrophoresis; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D004573:Electrolytes; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase