Abstract
The mechanism by which cells reduce cytoplasmic vanadium(V) (vanadate) to vanadium(IV) was investigated using the human red cell as a model system. Vanadate uptake by red cells occurs with a rapid phase involving chemical equilibration across the plasma membrane and a slower phase resulting in a high concentration of bound vanadium(IV).
The slow phase was inhibited in glucose-starved cells and restored upon addition of glucose indicating an energy requirement for this process. The time course of vanadium(IV) appearance (monitored by EPR spectroscopy of intact cells) paralleled the show phase of uptake indicating that this phase involves vanadium reduction. The reduction of intracellular vanadate to vanadium(IV) was nearly quantitative after 23 h. The intracellular reduction is not enzymatic, since a similar time course of vanadium reduction and binding to hemoglobin was observed when glutathione was added to a hemoglobin + vanadate solution in vitro. Vanadium(IV) bidning to hemoglobulin was reduced by addition of ATP, 2,3-diphosphoglycerate or EDTA, probably through chelation of the cation. The stability constant of the ATP-vanadium (IV) complex was determined to be 150 M
−1 at pH 4.9.
The time course of red cell vanadate uptake and reduction was followed in the concentration range in which approximately 60% inhibition of the (Na
+ + K
+)-ATPase is observed. It is concluded that vanadate is reduced by cytoplasmic glutathione in this concentration range and that the reduction explains the resistance of the (Na
+ + K
+)-ATPase to vanadium in intact cells.