Vanadate inhibits (Na⁺ + K⁺)ATPase by blocking a conformational change of the unphosphorylated form

Abstract

In the presence of extracellular K, intracellular orthovanadate is a potent inhibitor of (Na⁺ + K⁺)ATPase^1–3, and because this effect may have a physiological role^3–6, it is interesting to ask at which step in the enzyme cycle vanadate acts. In experiments with red cells and squid axons containing high concentrations of ATP, Beaugé and DiPolo^7,8 found that added vanadate modified the effects of extracellular K, or congeners of K, in a similar way to lowered ATP concentration. They suggested that vanadate might act by retarding the relatively slow, ATP-sensitive, conformational change that is thought to follow the hydrolysis of phosphoenzyme, when that hydrolysis is catalysed by extracellular K (ref. 9). There are several reasons for thinking that the same conformational change (E₂K → E₁K) is the rate-limiting step in the conversion of E₂K to E₁Na that occurs when sufficient Na is added to (Na⁺ + K⁺)ATPase preparations suspended in low-K media in conditions which preclude phosphorylation^10–14. The speed of the E₂K → E₁K change, in those circumstances, can be measured by the use of several different fluorescent techniques^10–12,15, and we report here experiments with a fluorescein-labelled enzyme to investigate the effects of vanadate. It turns out that, in the same conditions in which vanadate inhibits (Na⁺ + K⁺)ATPase activity, it greatly slows the rate of the conformational change E₂K → E₁K.