Chemical aspects of the influence of cobalt ions on ATPase activity

Abstract

The influence of Co2+ ions on the activities of Na+/K+-ATPase and Mg2+-ATPase, enzymes from rat brain synaptic plasma membrane, was studied. The aim of this study was to investigate the inhibition of both ATPases activities by exposure to cobalt ions as a function of experimentally added CoSO4. The "free" Co2+ concentrations in the reaction mixture were also calculated and discussed. CoSO4 induced a dose-dependent inhibition of both enzymes. The IC50 values of Co2+, as calculated from the experimental curves, were 168 mu M for Na+/K+-ATPase and 262 mu M for Mg2+-ATPase, and for the recalculated free Co2+ concentration 75.4 mu M for Na+/K+-ATPase and 136 mu M for Mg2+-ATPase. The obtained linear Dixon's plot for Na+/K+-ATPase implies equilibrium binding of cobalt with inhibitory sites on the enzyme. The kinetic parameters for both enzymes in presence and absence of CoSO4 were calculated from the experimental data. The results of the kinetic analysis show that inhibition of Na+/K+-ATPase induced by CoSO4 is non-competitive, and for Mg2+-ATPAse that there are two sites of different sensitivities or two different enzymes.

Ispitan je uticaj Co2+ jona na aktivnost Na+/K+ -ATPaze i Mg2+ -ATP-aze, enzima sinaptozomalne membrane mozga pacova. Cilj rada je bio da se ispita inhibicija aktivnosti oba enzima izazvana izlaganjem jonima kobalta kao funkcija eksperimentalno dodatog CoSO4. Takođe je izračunata i diskutovana "slobodna" koncentracija Co2+ u reakcionoj smeši. Utvrđeno je da kobalt inhibira enzime u koncentraciono zavisnom smislu. Vrednosti IC50 izračunate iz eksperimentalnih krivih su: 168 μM za Na+/K+ -ATP-azu i 136 μM za Mg2+ -ATP-azu. Linearan Dixon-ov plot za Na+/K+ -ATP-azu ukazuje na ravnotežno vezivanje kobalta. Izračunati su kinetički parametri oba enzima u prisustvu i odsustvu CoSO4. Inhibicija Na+/K+-ATP-aze izazvana CoSO4 je nekompetitivna, dok Mg2+ -ATP-aza ima dva mesta vezivanja različitog afiniteta ili pak dva različita enzima.