To characterize the location of glycolytic enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1-12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.GAPDH ͉ band 3 ͉ AE1 ͉ metabolon ͉ compartmentalization

Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane anion transport protein (band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a 1 H-13 C NMR technique for measuring metabolic fluxes in intact cells. The role of band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE-band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of band 3 in mediating oxygen-regulated metabolic transitions.erythrocyte ͉ glycolysis ͉ pervanadate ͉ NMR

Peroxiredoxin 2 (Prx2), the third most abundant cytoplasmic protein in red blood cells (RBCs), is involved in the defense against oxidative stress. Although much is known about Prx2 in healthy RBCs, its role in pathological RBCs remains largely unexplored. Here, we show that the expression and net content of Prx2 are markedly increased in RBCs from two mouse models of β-thalassemia (β-thal; Hbbth/th and Hbbth3/+ strains). We also demonstrate that the increased expression of Prx2 correlates with the severity of the disease and that the amount of Prx2 bound to the membrane is markedly reduced in β-thal mouse RBCs. To explore the impact of oxidative stress on Prx2 membrane association, we examined Prx2 dimerization and membrane translocation in murine RBCs exposed to various oxidants (phenylhydrazine, PHZ; diamide; H2O2). PHZ-treated RBCs, which mimic the membrane damage in β-thal RBCs, exhibited a kinetic correlation among Prx2 membrane displacement, intracellular methemoglobin levels, and hemichrome membrane association, suggesting the possible masking of Prx2 docking sites by membrane-bound hemichromes, providing a possible mechanism for the accumulation of oxidized/dimerized Prx2 in the cytoplasm and the increased membrane damage in β-thal RBCs. Thus, reduced access of Prx2 to the membrane in β-thal RBCs represents a new factor that could contribute to the oxidative damage characterizing the pathology.

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking ␣-spectrin, ankyrin, protein 4.2, protein 4.1, ␤-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3. (Blood. 2008;112:3900-3906)