Metabolic dependence of protein arrangement in human erythrocyte membranes. II. Crosslinking of major proteins in ghosts from fresh and ATP-depleted red cells.

We have investigated changes in membrane protein organization of ATPdepleted red cells by employing intermolecular crosslinking of the neighboring membrane proteins with glutaraldehyde or catalytic oxidation. After oxidative crosslinking, the complexes of crosslinked proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS PAGE) and treated with a reducing agent, dithiothreitol (DTT), which subsequently cleaved the intermolecular disulfide bonds. The individual protein components of the crosslinked complexes were subsequently identified by two-dimensional SDS PAGE. Catalytic oxidation of ghosts from both fresh and anaerobically ATPdepleted red cells produced crosslinked oligomers of spectrin (heterodimers, trimers, and tetramers), oligomers of band 3 (dimers, trimers, and tetramers), complexes of 2 + 4.9, and dimers of 4.1, 4.5, and 5 (actin). In addition, crosslinked ghosts from ATP-depleted cells, but not from fresh red cells, contained an additional large molecular weight (>106 daltons) complex that was enriched in spectrin, suggesting a rearrangement of spectrin in the membrane to closer contacts. Maintenance of intracellular ATP stores during anaerobic incubation prevented the formation of this complex after subsequent crosslinking. In contrast, the propensity of ghosts to form this complex after crosslinking was not reversed by restoration of ATP level or by EDTA extraction of membrane-bound calcium. In addition to reducible membrane protein complexes, introduction of calcium (>0.5 mM) into fresh red cells by ionophore A231 87 or into their ghosts produced a nonreducible large molecular weight polymer, which was shown by others to result from membrane protein crosslinking catalized by Ca2 ‘ -stimulated transglutaminase. This crosslinking is markedly decreased in ATP-depleted cells due to inactivation of the cytoplasmic transglutaminase during ATP depletion. Our data indicate that the organization of spectrin and other polypeptides in red cell membranes depends on intracellular ATP and calcium levels.