METABOLIC DEPENDENCE OF PROTEIN ARRANGEMENT IN HUMAN ERYTHROCYTE-MEMBRANES .1. ANALYSIS OF SPECTRIN-RICH COMPLEXES IN ATP-DEPLETED RED-CELLS

Abstract

The discocyte-echinocyte transformation and the decrease in deformability associated with red cell ATP depletion have been attributed to changes in the physical properties of spectrin and actin, membrane proteins located at the membrane-cytosol interface. The experiments reported here investigated the spontaneous formation of spectrin-rich complexes in human erythrocyte membranes, employing 2-dimensional SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis. Membranes of red cells depleted in ATP under aerobic conditions exhibited the following properties: an increase in components 4.5 and globin subunits; a spontaneous formation of heterodimers of spectrin 1 + 2 and spectrin 2 + component 4.9; and a large MW (> 106 daltons) protein complex with a high spectrin to band 3 ratio. These complexes were dissociated with dithiothreitol and were prevented by anaerobic incubation or the maintenance of red cell ATP and GSH [glutathione] levels with glucose, adenine and inosine. The complexes 1 + 2 and 2 + 4.9 were also seen in acetylphenylhydrazine-treated, glucose-6-phosphate dehydrogenase-deficient fresh erythrocytes that showed marked GSH depletion but preserved > 70% of the original ATP level. Membranes of these cells did not contain the > 106-dalton aggregate with a high sepctrin to band 3 ratio. The formation of the latter complex results from rearrangement of spectrin and other polypeptides in membranes of ATP-depleted red cells. Under aerobic conditions, the rearranged proteins undergo spontaneous intermolecular crosslinkings through disulfide couplings.