Characterisation of Phospho enol pyruvate Transport across the Erythrocyte Membrane

Phospho enol pyruvate was found to be transported across the erythrocyte membrane by a carrier‐mediated transport system. The transport of phospho enol pyruvate was competitively inhibited by inorganic phosphate ( K i = 24mM) and pyridoxal 5‐phosphate ( K i = 0.2 mM), whereas the transport was noncompetitively inhibited by L(+)‐lactate ( K i = 37 mM). Specific inhibitors for the inorganic anion transport system such as 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid and 4,4′‐dinitrostilbene‐2,2′‐disulfonic acid strongly inhibited the phospho enol pyruvate transport. The transport was irreversibly inhibited by treating erythrocytes with pyridoxal 5‐phosphate and NaBH 4 . Transport activities of Phosph enol pyruvate and inorganic phosphate in the treated cells were similary inhibited by pyridoxal 5‐phosphate depending on its concetrations. 4,4′‐Dinitrostilbene‐2,2′‐disulfonic acid protected both transport activities against the pyridoxal 5‐phosphate/NaBH 4 treatment. The major integral membrane protein, band 3, was preferentially labelled by treating erythrocytes with pyridoxal 5‐phosphate and NaB[ 3 H]H 4 . the radioactive incorporation into band 3 was confirmed by two‐dimensional gel electrophoresis combining isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the second dimension. Taken together, these results suggest that band 3 mediates the transport of phospho enol pyruvate as well as inorganic phosphate.