Dual Mechanisms of Allosteric Acceleration of the Na+,K+-ATPase by ATP

Abstract

Investigations of the E2 --> E1 conformational change of Na(+),K(+)-ATPase from shark rectal gland and pig kidney via the stopped-flow technique have revealed major differences in the kinetics and mechanisms of the two enzymes. Mammalian kidney Na(+),K(+)-ATPase appears to exist in a diprotomeric (alphabeta)(2) state in the absence of ATP, with protein-protein interactions between the alpha-subunits causing an inhibition of the transition, which occurs as a two-step process: E2:E2 --> E2:E1 --> E1:E1. This is evidenced by a biphasicity in the observed kinetics. Binding of ATP to the E1 or E2 states causes the kinetics to become monophasic and accelerate, which can be explained by an ATP-induced dissociation of the diprotomer into separate alphabeta protomers and relief of the preexisting inhibition. In the case of enzyme from shark rectal gland, the observed kinetics are monophasic at all ATP concentrations, indicating a monoprotomeric enzyme; however, an acceleration of the E2 --> E1 transition by ATP still occurs, to a maximum rate constant of 182 (+/- 6) s(-1). This indicates that ATP has two separate mechanisms whereby it accelerates the E2 --> E1 transition of Na(+),K(+)-ATPase alphabeta protomers and (alphabeta)(2) diprotomers.

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

“…Thus these data suggest that there are sites of phosphorylation in the N-terminus of the α -subunit that could play a role in the formation of Population #1. The Na +/ K + -ATPase in the kidney kinetically functions as a ( αβ ) 2 heterodimer [27–29]. However, these data do not exclude the possibility that at least some of the Na +/ K + -ATPase in the kidney plasma membrane could operate as a αβ protomer.…”

Cholesterol depletion inhibits Na+,K+-ATPase activity in a near-native membrane environment

“…Stopped-flow experiments were performed using an SF-61SX2 stopped-flow spectrofluorimeter (TgK Scientific, Bradford on Avon, UK) as described previously (32,54,81). All solutions were equilibrated to a temperature of 24°C before measuring the kinetics.…”

Membrane Electrochemistry: Electrochemical Processes in Bilayer Lipid Membrane

“…This assumption was confirmed by analysis of the noise level in ionic currents resulting from channel opening and closing. In this regards we should mentioned here that the structure of the Na and K channels differ from type to type of animal classes, recently from our study on the conformational changes of Na + ,K + -ATPase from some type of animal classes using stopped-flow techniques have revealed major differences in the kinetic mechanisms (and hence enzyme structure) of mammalians and non-mammalians enzymes [69,70], in the absence of ATP appear that the mammalian Na + ,K + -ATPase exists in a diprotomeric state ( )2, with protein-protein interactions between the -subunits causing an inhibition of the transition, while binding of ATP to any of the enzyme conformational states induced the dissociation of the diprotomer into separate protomers and relief the pre-existing inhibition, non-mammalians exhibits no effect of ATP binding on the enzyme at all concentrations indicating a mono-protomeric structure ( protomers).…”