Effects of Digoxin and Gitoxin on the Enzymatic Activity and Kinetic Parameters of Na⁺/K⁺-ATPase

Abstract

Inhibition of Na+/K+-ATPase activity from human erythrocyte membranes and commercial porcine cerebral cortex by in vitro single and simultaneous exposure to digoxin and gitoxin was investigated to elucidate the difference in the mechanism of the enzyme inhibition by structurally different cardiac glycosides. The drugs exerted a biphasic dose-dependent inhibitory effect on the enzyme activity in both tissues, supporting the existence of two sensitive Na+/K+-ATPase isoforms. The IC50 values for the low and high affinity isoforms were calculated from the inhibition curves using mathematical analysis. The Hill coefficient (n) fulfilled the relationship 1 < n < 3, suggesting cooperative binding of inhibitors to the enzyme. Kinetic analysis showed that digoxin and gitoxin inhibited Na+/K+-ATPase by reducing the maximum enzymatic velocity (Vmax) and Km, implying an uncompetitive mode of interaction. Both the isoforms were always more sensitive to gitoxin. The erythrocyte enzyme was more sensitive to the inhibitors in the range of low concentrations but the commercial cerebral cortex enzyme exerted a higher sensitivity in high inhibitors affinity concentration range. By simultaneous exposure of the enzyme to digoxin and gitoxin in combinations a synergistic effect was achieved by low inhibitor concentrations. An antagonistic effect was obtained with erythrocyte membrane enzyme at high inhibitors concentration.

Na+,K+-ATPase as the Target Enzyme for Organic and Inorganic Compounds

“…The Na + ,K + -ATPase used in the inhibition studies could be isolated from different sources (rat brain, dog kidney, porcine cerebral cortex, human blood) [48, 52, 54, 67]. Each choice of the Na + ,K + -ATPase source, i.e.…”

Pharmacological Targeting of the Mitochondrial Phosphatase PTPMT1

“…Furthermore, in our study characterizing the IC 50 for inhibition of PTPMT1 by alexidine dihydrochloride, we noted a Hill coefficient of 2, suggesting cooperativity of binding of the inhibitor to the enzyme. This cooperativity might be achieved where binding of the inhibitor to one site on the enzyme facilitates binding to a second site on the same molecule (Krstic et al, 2004) or where the enzyme functions as dimer and binding of one enzyme molecule by the inhibitor facilitates binding of the second enzyme molecule. Considering these observations together with the striking binate structure of alexidine dihydrochloride, and the requirement for the binate structure for effective inhibition of the enzyme, we speculate that the compound works by binding sites on each of two molecules of an enzyme dimer, which are only available in close proximity after substrate binding.…”

Influence of decavanadate on rat synaptic plasma membrane ATPases activity

“…Na + /K + -ATPase is a cell membrane located enzyme that establishes and maintains the high internal K + and low internal Na + concentrations, characteristic and essential for normal cellular activities of most animal cells (Vasilets and Schwarz 1993;Rodriguez de Lores Arnaiz and Pena 1995). The activity of this enzyme is very sensitive to the presence of some metal ions and organic compounds of various structures, especially some drugs and pesticides (Blasiak 1995;Krstić et al 2004Krstić et al , 2005. PMCA is a fine tuner of cytosolic calcium concentration in excitable cells, while the calcium pump is the sole system responsible for extrusion of calcium ions outside nonexcitable cells (Zylinska and Soszynski 2000).…”