Rate Limitation of the Na+,K+-ATPase Pump Cycle

Abstract

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

“…10 shows traces of the E 1 3Na-E 2 P and E 2 (2K)ATP-E 1 (3Na)ATP interconversions for preparations made with SOPS/cholesterol alone or with brain PE or with SM/cholesterol, and Table 5 shows the data for fitted rate constants. As described previously (47), the E 1 -E 2 P transition showed a rapid major phase and minor slow phase. Compared with the SOPS sample (k 1 ϭ 131 Ϯ 9 s Ϫ1 ), the rapid phase (k 1 ) was clearly accelerated by brain PE (270 Ϯ 10 s Ϫ1 ) but was unaffected by SM/cholesterol (162 Ϯ 12 s Ϫ1 ) (Fig 10A).…”

“…), EP levels (ϳ5 nmol/mg), kinetic properties (K 0.5 Na , K 0.5 K , and vanadate and ouabain inhibition; Tables 3 and 4), rates of conformational changes (Table 5), and stability in different conformations (8,41,47,57). The similar effect of the PE on Na,K-ATPase activity of all three isoforms (Fig.…”

Regulation of Na,K-ATPase by PLMS, the Phospholemman-like Protein from Shark

“…The preparations were assayed using the following experimental approaches: (i) measurements of the Na ϩ -, K ϩ -, and ATP-activation of Na,K-ATPase catalytic activity at V max conditions, (ii) activation of the Na-ATPase reaction by K ϩ at low ATP concentration to measure the K ϩ deocclusion pathway, (iii) measurements of the Na ϩ activation curve at low ATP concentration to probe effects on the Na ϩ -binding affinity, (iv) measuring the vanadate sensitivity of control and PLMS-truncated preparations to probe the E 1 /E 2 equilibrium, and (v) measurements of the low affinity ATP-supported transition E 2 (K) 3 E 1 (Na)ATP and the following phosphorylation reaction pathway leading to E 2 -P using time resolved fluorescence measurements. The latter reactions include the major rate-limiting steps of the Na,KATPase catalytic cycle under physiological conditions (42,50,51).…”

“…That the former could be the case was indicated by measurements of the main rate-limiting E 2 (K) 3 E 1 NaATP reaction at physiological conditions, including the low affinity ATP binding (42,50,51). This reaction can be measured by stopped-flow fluorescence using the potential sensitive styryl dye RH421 (42).…”

Mechanism of the Rate-Determining Step of the Na+,K+-ATPase Pump Cycle

“…The kinetics of the conformational transition from E 2 to E 1 can be studied by rapidly mixing the enzyme with a sufficient excess of Na + ions over K + , which leads to a conversion of the enzyme into the E 1 (Na + ) 3 state. Such experiments were first carried out by Karlish and Yates (5), who used changes in the intrinsic tryptophan protein fluorescence to monitor the kinetics of the conformational change.…”