Modulation of red cell glycolysis: interactions between vertebrate hemoglobins and cytoplasmic domains of band 3 red cell membrane proteins

Abstract

Several vital functions/physical characteristics of erythrocytes (including glycolysis, the pentose phosphate pathway, ion fluxes, and cellular deformability) display dependence on the state of hemoglobin oxygenation. The molecular mechanism proposed involves an interaction between deoxyhemoglobin and the cytoplasmic domain of the anion-exchange protein, band 3 (cdB3). Given that band 3 also binds to membrane proteins 4.1 and 4.2, several kinases, hemichromes, and integral membrane proteins, and at least three glycolytic enzymes, it has been suggested that the cdB3-deoxyhemoglobin interaction might modulate the pathways mediated by these associated proteins in an O(2)-dependent manner. We have investigated this mechanism by synthesizing 10-mer peptides corresponding to the NH(2)-terminal fragments of various vertebrate cdB3s, determining their effects on the oxygenation reactions of hemoglobins from the same and different species and examining binding of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase to the erythrocytic membrane of mouse erythrocytes. The cdB3 interaction is strongly dependent on pH and the number of negative and positive charges of the peptide and at the effector binding site, respectively. It lowers the O(2) association equilibrium constant of the deoxygenated (Tense) state of the hemoglobin and is inhibited by magnesium ions, which neutralize cdB3's charge and by 2,3-diphosphoglycerate, which competes for the cdB3-binding site. The interaction is stronger in humans (whose erythrocytes derive energy predominantly from glycolysis and exhibit higher buffering capacity) than in birds and ectothermic vertebrates (whose erythrocytes metabolize aerobically and are poorly buffered) and is insignificant in fish, suggesting that its role in the regulation of red cell glycolysis increased with phylogenetic development in vertebrates.

Carbon Monoxide Signaling in Human Red Blood Cells: Evidence for Pentose Phosphate Pathway Activation and Protein Deglutathionylation

“…Table 1 also shows that G6PDH and 6PGDH activities in cell lysates of CO-treated RBCs were increased by about 22% and 24%, respectively, versus air-equilibrated samples. Notably, in hypoxic conditions, the activities of these enzymes were significantly inhibited by about 21% and 26%, respectively, due to enhancement of glycolysis and reduction of PPP flux (23,55). Collectively, these results suggest that CO does not trigger glycolysis, as previously reported (21,23), but boosts PPP, presumably to generate NADPH.…”

Functional Properties of Neuroglobin and Cytoglobin. Insights into the Ancestral Physiological Roles of Globins

“…The proteins that until now have been shown to interact with Ngb in vitro are not normally involved in regulatory cascade responses as would be expected if Ngb was involved in signal transduction pathways. An alternative requirement would be oxygenationlinked interactions with molecules that are implicated in specific regulated processes -as exemplified by vertebrate Hbs that may regulate glycolysis by competing with glycolytic enzymes for binding to band 3 red cell membrane proteins (47).…”

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

“…For each individual specimen, Hb isoform composition was characterized by means of alkaline PAGE and/or thin layer isoelectric focusing (PhastSystem, GE Healthcare Biosciences). Depending on the species, the HbA and HbD isoforms were separated by fast protein liquid chromatography (FPLC), DEAE anion-exchange chromatography, CM-Sepharose cation-exchange chromatography, and/or preparative electrofocusing, as described previously (11,36,43,44). The separate isoHbs were further stripped of organic phosphate and other ions by passing the samples through a mixed bed resin column (MB-1 AG501-X8; Bio-Rad) using FPLC.…”