Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

Abstract

The MgATP complex analogue cobalt-tetrammine-ATP [CO(NH~)~ATP] inactivates (Na' + K ')-ATPase at Despite the inactivation of (Na' + K+)-ATPase by C O ( N H~)~A T P from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 pM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na' + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the El and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na' + K +)-transport.Active transport of sodium and potassium through cellular membranes catalyzed by (Na' + K+)-ATPase is accompanied by conformational changes of the enzyme protein [I -41. High-and low-affinity ATP binding sites and a nonhyperbolic ATP saturation curve of the catalytic activity seem to be an expression this [5 -1 I]. While binding of ATP to the high-affinity ATP binding site appears to be essential for the Correspondence to G.

Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants

“…This agrees with studies of radiation inactivation (Briskin, 1990) and chemical modification (Brauer et al, 1992) that indicate a functional unit with a molecular mass of approximately 200 kD. The existence of a dimeric ensemble has been proposed for the mammalian Na+,K+-and Ca2+-and funga1 H+-ATPases (Goffeau and Slayman, 1981;Brooker and Slayman, 1983a, 198313;Bowman, 1983;Koland and Hammes, 1986;Scheiner-Bobis et al, 1987;Andersen, 1989;Norby and Jensen, 1989;Serpersu et al, 1990). That idea, with the addition of interacting monomers, can explain our results.…”

Shift to the Na+ from of Na+/K+‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH3)4ATP

“…As expected from the findings of an unaltered capacity of the high-affinity ATP-binding site [20,211 eosin, a fluorescent probe for the high-affinity ATP-binding site [22,351 is also recognized by the Co(NH,),ATP-inactivated enzyme (Fig. 4).…”

“…We reported recently, that the stable MgATP complex analogue Co(NH,),ATP inactivates Na+/K+-ATPase slowly by forming a relatively stable complex with the enzyme [20,211. The conformational state recognizing and containing Co(NH,),ATP has been proposed to be the E2 conformational state, because the ATP analogue binds to a lowaffinity ATP-binding site.…”

“…The interaction of the treated Na+/K+-ATPase with eosin was studied. [20]. To fortify this conclusion we furthermore tested a partial activity of the enzyme, namely the K +-activated p-nitrophenylphosphatase [l, 2, 30 -321.…”

“…This sometimes rather indirect data supports the concept of a cooperation of a$ protomers and raises the question how the catalytic c( subunits interact [7 -191. We recently showed that the MgATP complex analogue C O ( N H~)~A T P binds to the low-affinity ATP-binding site, which is found in the Ez conformational state [20, 211. In contrast to the expectations from the Albers-Post model, however, the capacity of the high-affinity ATP-binding site remained unaltered and the inactive enzyme was still able to phosphorylate the a subunit from the high-affinity ATP-binding site which is dependent on Na+ [20, 211.…”