Mechanism of Mg2+ Binding in the Na+,K+-ATPase
AUTOR(ES)
Pilotelle-Bunner, Anne
FONTE
The Biophysical Society
RESUMO
The Mg2+ dependence of the kinetics of the phosphorylation and conformational changes of Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na+)3 state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg2+ to saturating values of 195 (± 6) s−1 and 54 (± 8) s−1 for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (Kd) of Mg2+ interaction with the E1ATP(Na+)3 state was estimated as 0.069 (± 0.010) mM. This is virtually identical to the estimated value of the Kd of Mg2+-ATP interaction in solution. Within the enzyme-ATP-Mg2+ complex, the actual Kd for Mg2+ binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2711396Documentos Relacionados
- Glucostatic regulation of (+)-[3H]amphetamine binding in the hypothalamus: correlation with Na+,K+-ATPase activity.
- Multiple genes encode the human Na+,K+-ATPase catalytic subunit.
- Regulation of endocytic pH by the Na+,K+-ATPase in living cells.
- Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation
- Monoclonal antibodies to rat Na+,K+-ATPase block enzymatic activity.