Pyruvate transport into inside-out vesicles isolated from human erythrocyte membranes.

Abstract 1. 1. A method has been developed for measuring the facilitated diffusion of pyruvate anions across the isolated human erythrocyte membrane. Ghosts were resealed in the presence of an excess of lactate dehydrogenase and NADH so that the influx of pyruvate could be followed by a continuous spectrophotometric recording of the oxidation of NADH (Rice, W.R. and Steck, T.L. (1976) Biochim. Biophys. Acta 433, 39–53). This approach has now been extended to sealed inside-out vesicles derived from the same membrane. 2. 2. Transport across inside-out and right-side-out membranes was similar in certain respects. In both cases, the influx of pyruvate was greatly stimulated by increasing the ionic strength in the external medium. Salts of transported anions (such as chloride) but not non-transported anions (such as malate or acetate) were inhibitory at high concentrations, presumably a competitive effect. The non-competitive inhibitors of anion flux, salicylate, 4-acetamido-4′-isothiocyanato-2,2′-stilbene disulfonate (SITS) and 1-fluoro-2,4-dinitrobenzene, reduced pyruvate transport from both sides of the membrane. 3. 3. Characteristic asymmetries in the transport of pyruvate were noted. Probenecid was found to act only at the cytoplasmic surface, although it inhibited flux in both directions. Mixtures of o- phenanthroline and CuSO 4 catalyzed the disulfide cross-linking of band 3, the purported anion transport protein, only at the membrane's cytoplasmic surface. This treatment stimulated pyruvate flux into resealed ghosts, but inhibited flux into inside-out vesicles. 4. 4. The binding of glyceraldehyde-3-phosphate dehydrogenase and aldolase to the cytoplasmic surface of the membrane did not significantly alter pyruvate flux in either direction, even though these enzymes are known to associate specifically with band 3. Proteolytic excision of the cytoplasmic domain of band 3, amounting to approximately 40% of its mass, also had no effect on pyruvate flux.