Characterisation of Phosphoenolpyruvate Transport across the Erythrocyte Membrane

Abstract

Phosphoenolpyruvate was transported across the [human] erythrocyte membrane by a carrier-mediated transport system. The transport of phosphoenolpyruvate was competitively inhibited by inorganic P (Ki = 24 mM) and pyridoxal 5-phosphate (Ki = 0.2 mM), whereas the transport was noncompetitively inhibited by L(+)-lactate (Ki = 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 phosphoenolpyruvate transport. The transport was irreversibly inhibited by treating erythrocytes with pyridoxal 5-phosphate and NaBH4. Transport activities of phosphoenolpyruvate and Pi in the treated cells were similarly inhibited by pyridoxal 5-phosphate depending on its concentrations. 4,4''Dinitrostilbene-2,2-disulfonic acid protected both transport activities against the pyridoxal 5-phosphate/NaBH4 treatment. The major integral membrane protein, band 3, was preferentially labeled by treating erythrocytes with pyridoxal 5-phosphate and NaB[3H]H4. The radioactive incorporation into band 3 was confirmed by 2-dimensional gel electrophoresis combining isoelectric focusing in the 1st dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis in the second dimension. Band 3 mediates the transport of phosphoenolpyruvate as well as Pi.