Rate Determination in Phosphorylation of Shark Rectal Na,K-ATPase by ATP: Temperature Sensitivity and Effects of ADP

Abstract

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na(+) was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E(2)-P, which follows formation of E(1)-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of approximately 3.1 x 10(7) M(-1) and 1. 8 x 10(5) M(-1), respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E(1)-P followed by a conformational change to E(2)-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K(+) the highly fluorescent E(2)-P is slowly transformed into the "K(+)-insensitive" E'(2)-P subconformation forming a side branch of the main cycle.

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

“…At saturating substrate concentrations the rate-limiting steps are the E 2 3 E 1 transition associated with low affinity ATP-supported K ϩ deocclusion and binding of cytoplasmic Na ϩ (51) and the E 1 ϳ P 3 E 2 -P transition, at least at temperatures below 15°C (58). Indeed, detailed investigations of the partial reactions showed that some steps along the K ϩ deocclusion pathway E 2 (K 2 ) 3 E 1 are accelerated after PLMS truncation (Fig.…”

“…The Phosphorylation/Dephosphorylation Reactions-The phosphorylation reaction was investigated by stopped-flow measurements using the membrane probe RH421 (41,58). This styryl dye partitions into the membrane containing Na,KATPase and is sensitive to the formation of E 2 -P.…”

“…12A show the formation of E 2 -P from mixing of the enzyme in the presence of 30 mM Na ϩ with 3 mM MgATP and thus represents the reactions E 1 Na 3 E 1 NaATP 3 E 1 ϳ P 3 E 2 -P. In shark Na,K-ATPase the initial phosphoryl transfer and formation of E 1 ϳP are faster than the formation of E 2 -P, at least at temperatures below 15°C (58). Thus, the stopped-flow fluorescence reaction mainly measures the rate of the E 1 ϳ P 3 E 2 -P reaction.…”

Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

“…During the recent years the transport cycle [1,3,27] has been broken down into a series of defined reaction steps and their kinetic properties were investigated and characterized [5,10,11,15,17,40]. In the case of the sodium transport branch the reaction sequence, 3 Na + cyt + E 1 → Na 3 E 1 → ͑Na 3 ͒E 1 -P → P-E 2 ͑Na 3 ͒ → P-E 2 ͑Na 2 ͒ + Na + → P-E 2 + Na + ext , is able to explain all experimental data available.…”

Assignment of charge movements to electrogenic reaction steps of Na,K-ATPase by analysis of salt effects on the kinetics of charge movements

“…Recent extensive studies have led to substantial progress in the understanding of the transport mechanism of the ion pump (Apell and Karlish 2001;Jùrgensen 1992). Detailed information on the kinetics of the active transport mechanism was obtained by electrical and optical studies, which allowed the identi®ca-tion of the electrogenic reaction steps of the transport cycle and the determination of their characteristic parameters (Clarke et al 1998;Cornelius 1999;Heyse et al 1994;Holmgren et al 2000;Sokolov et al 1998a;Wuddel and Apell 1995). Electric measurements can be performed in stationary and non-stationary modes.…”