Abstract
The fate of vanadate (+5 oxidation state of vanadium) taken up by the red cell was studied using EPR spectroscopy. The appearance of an EPR signal indicated that most of the cytoplasmic vanadate is reduced to the +4 oxidation state with axial symmetry characteristic of vanadyl ions. The signal at 23 degrees C was characteristic of an immobilized system indicating that the vanadyl ions in the cytoplasm are associated with a large molecule. [48V]Vanadium eluted with hemoglobin when the lysate from Na3[48V[O4-treated red cells was passed through a Sephadex G-100 column and rabbit anti-human hemoglobin serum caused a hemoglobin-specific precipitation of 48V when added to the red cell lysate. Both results indicate that hemoglobin is the protein which binds cytoplasmic vanadyl ions. However, neither sodium vanadate nor vanadyl sulfate bound to purified hemoglobin in vitro. Finally, transient kinetics of vanadyl sulfate interaction with the sodium-and potassium-stimulated adenosine triphosphatase showed that the +4 oxidation state of vanadium is less effective than the +5 oxidation state in inhibiting this enzyme. These results indicate that oxidation-reduction reactions in the cytoplasm are capable of relieving vanadate inhibition of cation transport.
Highlights
+4 oxidation state with axial symmetry characteristic of vanadyl ions
The spectrum observed is characteristic of vanadium in the +4 oxidation state indicating reduction of the vanadate ion has occurred
Addition of 300 pM dinitrostilbene disulfonate (which inhibits vanadate uptake by red cells [8]) to the suspension prior to vanadate addition lowered the amplitude of the EPR signal by 40% showing that vanadate must enter the cell for reduction to occur
Summary
Red cells were freshly prepared from L. Cantley’s blood as previously described [8]. Sodium vanadate and vanadyl sulfate were from Fisher Scientific. The (Na,K)-ATPase was prepared from dog kidneys by the procedure of Jorgensen [13]. R4*VlVanadvl chloride was from Amersham and was converted to vanadate as previously described [9]. EPR spectra were recorded with a Varian E-9 spectrometer operating at x-band frequencies. Studies at room temperature were done in the Varian flat aqueous sample cell, and at 77 K in a quartz liquid nitrogen Dewar and 3-mm quartz sample tubes. Spectrometer fields and frequencies were measured with a Varian model F-8A flux meter and a HewlettPackard model 540-B transfer oscillator, respectively, each tied to a Hewlett-Packard model 5240-L frequency counter. Spectral integrations were performed with a Fabri-Tek (Nicolet) model 1072 instrument computer
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