Hemoglobin-spectrin interaction under oxidative conditions.
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Cross-Linking Reagents↗
Biomedical subjects
N Sauberman
Publications and source records attributed to N Sauberman.
Irreversible spectrin-haemoglobin crosslinking in vivo: a marker for red cell senescence.
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Centrifugation, Density Gradient↗
Spectrin-haemoglobin crosslinkages associated with in vitro oxidant hypersensitivity in pathologic and artificially dehydrated red cells.
Protein crosslinkages are apparent at 215 000-250 000 daltons in electrophoregrams of membranes from hydrogen peroxide treated erythrocytes (British Journal of Haematology, 48, 435, 1981). Hydrogen peroxide is also capable of inducing crosslinkages of identical molecular weights in stage I and II (red) ghosts and in a mixture of purified spectrin and haemoglobin, but not in white ghosts or in either spectrin or haemoglobin alone. Autoradiographic studies using 14C-methaemoglobin and 32P-spectrin confirm the involvement of spectrin and haemoglobin in this reaction. The alpha chains of both proteins are more reactive than the corresponding beta chains: 3 times more reactive in the case of spectrin and 10 times more reactive in haemoglobin. The reaction is almost totally inhibited by NaCN and partially inhibited by N-ethylmaleimide. Direct addition of malondialdehyde to a spectrin-haemoglobin mixture does not result in protein crosslinkage. Metabolic depletion (40 h), in vivo ageing and sucrose dehydration of fresh, normal cells enhance the reaction considerably, whereas in vitro rehydration of xerocytes normalizes their H2O2 sensitivity.
Erythrocyte Membrane↗
Altered red blood cell surface area in hereditary xerocytosis.
Hereditary xerocytes appear larger than normal red cells in scanning electron micrographs and exhibit a higher ghost packing volume. The major chemical components--protein, phosphorus, cholesterol and sialic acid--are increased uniformly, as are all polypeptides visible on gel electrophoresis patterns of xerocyte membranes. These data are consistent with a xerocyte surface area 15 to 25% above normal. Certain clinical anomalies common to this disorder, including unexpectedly low reticulocyte count and 2,3-diphosphoglycerate level, are discussed in the light of the present findings.
Cholesterol↗
Red cell membrane response to hydrogen peroxide-sensitivity in hereditary xerocytosis and in other abnormal red cells.
Osmotically resistant red cells associated with some haemolytic anaemias, including hereditary xerocytosis, sickle-cell disease and beta thalassaemia minor, are more sensitive than normal red cells to exogenous in vitro hydrogen peroxide (H2O2). This sensitivity is manifested by a rapid loss of intracellular potassium, shape change, protein aggregation, and methaemoglobin formation at lower concentrations of H2O2 (225 microM) than are required to induce similar effects in normal red cells (450 microM). Malonyldialdehyde (MDA) formation occurs later than the other effects and can be inhibited by the antioxidant, butylated hydroxytoluene (BHT), without affecting protein aggregation or potassium leak. Incubation of normal red cells directly with MDA induces protein aggregation, but only after 1 h of incubation. Although nystatin-sucrose treated normal cells which are dehydrated with altered cation content, and therefore osmotically resistant, do not display abnormal H2O2 hypersensitivity as manifested by excessive potassium permeability, they do show an increase in methaemoglobin formation and protein aggregation similar to xerocytes. These data indicate that membrane protein cross-linking occurring immediately following H2O2 exposure seems independent of either the sulfhydryl or MDA mechanisms, and that the membrane permeability of the abnormal red cells predisposes them to oxidative damage.
Anemia, Hemolytic, Congenital↗
Contribution of whole cell and cytoplasmic polypeptides to apparent red cell membrane alterations.
We have compared densitometric tracings of whole cell, cytoplasmic and membrane polypeptide electrophoretic patterns in an attempt to distinguish atypical partitioning from intrinsic membrane polypeptide changes occurring as a result of reticulocyte enrichment, metabolic depletion, N-ethylmaleimide treatment and hereditary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in tary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in the partitioning of polypeptides between membranes and supernatant (cytoplasm) at hemolysis. The power of this approach in resolving the sources of apparent red cell membrane protein alterations is demonstrated in studies with hereditary xerocytes. Suggested altered partitioning of these cells described earlier (Sauberman, N., Fortier, N.L., Fairbanks, G., O'Connor, R.J. and Snyder, L.M. (1979) Biochim. Biophys. Acta 556, 292-313) is further documented and found to be unrelated to the younger cell population or slight metabolic depletion that occurs during the washing of xerocytes prior to hemolysis.
Cytoplasm↗
Red cell membrane in hemolytic disease. Studies on variables affecting electrophoretic analysis.
Significant alterations in the spectrin: band 3 and band 4.1a: band 4.1b ratios and an occasional decrease in the peak height of band 4.2 with respect to band 4.1 were found in electrophoretic patterns of red cell membranes from patients with hereditary xerocytosis. Electrophoretic comparison of whole cell, cytoplasm and membrane polypeptides implied that atypical partitioning at hemolysis could account for some, but not all, of the alterations seen in membrane patterns of xerocytes. A decrease in band 4.2 peak height as well as a variation in the profile of band 3 were produced in controls by specific manipulations of the electrophoresis protocol. Metabolic depletion of normal cells produced the type of alterations in bands 3 and 4.1 found in xerocyte membranes, whereas Heinz body production, addition of calcium to the hemolysis buffer and incubation of membranes in detergent under conditions designed to promote proteolysis did not. The presence of a higher peak height of band 4.1b with respect to that of band 4.1a in membranes of patients with various other red cell disorders correlated with an increase in the percentage of reticulocytes in peripheral circulation. The appearance of both band 3 and 4.1 abnormalities in the patterns of control cells which had been enriched in young cells by density gradient centrifugation suggested that these alterations in hemolytic disease are related to the predominance of young cells in the population.
Adenosine Triphosphate↗
Fragmentation and myelin formation in hereditary xerocytosis and other hemolytic anemias.
Erythrocytes from a heterogeneous group of hemolytic anemias have been found to release acetylcholinesterase-enriched fragments and show myelin forms during ATP depletion in vitro. The highest amount of fragmentation was found in hereditary spherocytosis and xerocytosis, two inherited membrane defects. Our data suggest ATP depletion plays a role in producing fragmentation or myelin forms. The addition of external CaCl2 1 mM had no effect on the degree of fragmentation. However, propranolol hydrochloride, a cationic anesthetic that does not prevent ATP depletion, inhibited fragmentation and the appearance of myelin forms in both hereditary spherocytes and xerocytes. A more detailed study of the xerocyte fragments showed that they had the same protein composition as those from normal red cells, primarily integral membrane proteins and glycoproteins. The red cells from patients with PNH and G6PD deficiency had the shortest survival in vivo (51Cr) and produced the smallest amount of fragmentation and myelin forms in vitro, whereas xerocytosis with only mild to moderate hemolysis in vivo was associated with the highest amount of myelin forms and membrane fragments in vitro.
Acetylcholinesterase↗