Membrane protein organization in ATP-depleted and irreversibly sickled red cells.

Abstract

AbstractIt has been proposed that the spectrin‐actin submembrane network participates in control of red cell shape and deformability. We have examined ATP‐ and calcium‐dependent changes in organization of spectrin in the membrane employing cross‐linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO4, O‐phenanthroline) intermolecular disulfide couplings and two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis.Cross‐linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP‐depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW > 1 × 106 selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross‐linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP‐dependent discocyte‐echinocyte transformation. This suggests that, in ATP‐depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte‐echinocyte transformation.The introduction of greater than 0.5 mM Ca++ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP‐depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross‐linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP‐depleted red cells and nonreducible in fresh, Ca++ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape.Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca‐ and ATP‐dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin‐rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5–10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero‐echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs.These data provide evidence of altered spectrin organization in membranes of ATP‐depleted, calcium‐enriched red cells in vitro and in vivo.