Abstract
The exact chemical composition of the red blood cell (RBC) membrane may vary depending on the methods used to isolate the membrane. We provide evidence here that RBC membrane can be fractionated by differential centrifugation and/or density gradient centrifugation into two distinct types, designated as ‘heavy membrane’ (HM) and ‘light membrane’ (LM). The amount of LM is twice that of HM on a per cell basis. HM and LM differ in some biochemical and electrophoretic properties. The total activities of Na+, K+ - and Ca2+- ATPases, superoxide dismutase, glutathione peroxidase, catalase and glucose-6-phosphate and 6-phosphogluconate dehydrogenase are significantly higher in LM than HM on a per cell basis. While there is no significant difference in the specific activity of other enzymes between the two membranes, the specific activity of Ca2+-ATPase is significantly higher in HM, whereas Na+, K+-ATPase activity is higher in LM. There is a remarkable difference in the distribution of major ghost polypeptides between these two membranes. Component I of spectrin, component III and a protein with mol. wt. of 165 KDa are present in smaller amounts, whereas component II of spectrin and proteins with mol. wt. of 145, 84 and 76 KDa, respectively, are present in higher amounts in HM than LM. Some proteins such as band 4.1, 48 and 46 KDa are present only in LM, whereas some proteins with mol. wt. of 96, 78 and 43 KDa, respectively are present only in HM. It has been confirmed that these two membranes are not representatives of either (a) right side-out vs. inside out vesicles, or (b) open vs. sealed membranes. Thus HM and LM are two distinct membrane fractions. It is suggested that some part of the membrane is denser than other parts, and during hemolysis of RBCs, the rbc membrane is spliced resulting in two populations, dense and light.
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References
Weed RI, LaCelle PL: The Red cell membrane: Structure and function. In: T.J. Greenwalt and G.A. Jamieson (eds). J.B. Lippincott Co., Philadelphia, 1969, pp. 318–351
Ponder E: The Cell. In: J. Brachet and A.E. Mirsky (eds). Vol. 11, Academic Press, New York, 1961, pp. 1–20
Dodge JT, Mitchell C, Hanahan DJ: The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100: 119–130, 1963
Steck TL, Weinstein JH, Strauss JH, Wallach DFH: Inside-out red cell membrane vesicles: Preparation and purification. Science (Wash D.C.) 168: 255–257, 1970
Moore GL, Koeholaty WF, Cooper JL, Gray JL and Robison SL: A proteinase from human erythrocyte membrane. Biochim Biophys Acta 212: 126–133, 1970
Steck TL, Kant JA: Preparation of impermeable ghosts and inside-out vesicles from human erythrocyte membranes. In: S. Fleischer and L. Packer (eds). Methods Enzymology. Vol. 31, Biomembranes, part A, Academic Press, New York, N.Y, 1974, pp. 172–180
Beutler E, West Duarte C, Blume KG: The removal of leukocytes and platelets from whole blood. J Lab Clin Med 88: 328–333, 1976
Burton GW, Ingold KU, Thompson KE: An improved procedure for the isolation of ghost membranes from human red blood cells. Lipids 16: 946, 1981
Skipi MO, Das SK: The localization of cholinephosphotransferase in the outer membrane of guinea pig lung mitochondria. Biochim Biophys Acta 899: 35–43, 1987
Hawk PB, Oser BL, Summerson WH: Practical Physiological Chemistry, 13th edn., Mc Graw-Hill, London, 1954, pp. 617–619
Peterson GL: A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 346–356, 1977
Hyland K, Voisin E, Banoun H, Auclair C: Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-gene rating system. Anal Biochem 135: 280–287, 1983
Flohe L, Gunzler WA: Assays of glutathione peroxidase. Meth Enzymol 105: 114–121, 1984
Das SK, Nair CR: Superoxide dismutase, glutathione peroxidase, catalase and lipid peroxidation of normal and sickled erythrocytes. Br J Haematol 44: 87–92, 1980
Beutler E: Methods in haematology: In red cell metabolism. In: E. Beutler (ed). Vol. 16, Churchill, Livingstone, 1986, pp. 49–72
Robinson JD: Kinetic studies on a brain microsomal adenosine triphosphatase. Evidence suggesting conformational changes. Biochemistry 6: 3250–3258, 1967
Nandi J, Ray TK, Sen PC: Studies of gastric Ca2+-stimulated adenosine triphosphatase. 1. Characterization and general properties. Biochem Biophys Acta 646: 457–464, 1981
Laemmli UK: Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature (London) 227: 680–685, 1970
Oakley BR, Kirsch DR, Morris NR: A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gel. Anal Biochem 105: 361–363, 1980
Kondo T, Dale GL, Beutler E: Simple and rapid purification of inside-out vesicles from human erythrocytes. Biochim Biophys Acta 602: 127–130, 1980
Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods, 2nd edn, Grune & Stratton, New York, 1975
Jain SK: Evidence for membrane lipid peroxidation during the in vivo aging of human erythrocytes. Biochim Biophys Acta 937: 205–210, 1988
Morrison WL, Neurath H: J Biol Chem 200: 39–51, 1953
Fairbanks G, Steck TL, Wallach DFH: Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10: 2606–2617, 1971
Mukherjee S, Hinds JE, Olson EJ, Weaver CG, Childs KR, Khan QA, Hardy RE, Das SK: The effects of physical stress on calcium and peroxide scavengers in normal and sickle cell rbc. FASEB J 2: A1224, 1988
Dzandu JK, Deh ME, Barratt DL, Wise GE: Detection of erythrocyte membrane proteins, sialoglycoproteins, and lipids in the same polyacrylamide gel using a double staining technique. Proc Natl Acad Sci 81: 1733–1737, 1984
Sheetz MP: in Erythocyte mechanics and blood flow, Alan R. Liss, Inc., N.Y., 1980, pp. 1–13
Marchesi VT, Furthmayr H: The red cell membrane. Ann Rev Biochem 45: 667–698, 1976
Litman D, Chen JH, Marchesi VT: J. Supramol Struct 8: 209a, 1978
Bennett V, Barton D: Selective association of spectrin with cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P) spectrin. J Biol Chem 252: 2753–2763, 1977
Liu SC, Palek J: In Erythrocyte Mechanics and Blood Flow, Alan R. Liss, Inc. N.Y., 1980, pp. 15–29
Sheetz MP, Singer SJ: On the mechanism of aTP-induced changes in human erythrocyte membranes. 1. The role of the spectrin complex. J Cell Biol 73: 638–646, 1977
Schrier SL: Shape changes and deformability in human erythrocyte membranes. J Lab Clin Med 110: 791–797, 1987
Juliano RL: The proteins of the erythrocyte membrane. Biochim Biophys Acta 300: 341–378, 1973
Van Deenen LLM, Degier J: The red blood cell. In: D.M. Surgenor (ed). Academic Press, New York, 1974, pp. 147–211
Platt D, Rieck W: Blood cells, rheology and aging. In: D. Platt (ed). Springerverlag, N.Y., 1985. pp. 29–41
Clark MR, Shohet SB: Red cell senescence. Clin Haematol 14: 223–257, 1985
Hochstein P, Jain SK: Association of lipid peroxidation and polymerization of membrane proteins with erythrocyte aging. Fed Proc 40: 183–188, 1981
Mukherjee S, Olson E, Hall LC, Hinds JE, Das SK: Effects of physical stress on membrane-bound ATPases, calcium and deformability in sickle cell trait rbc. 14th Annual Sickle Cell Center Conference: Strategies for Therapy and Care in Sickle Cell Disease, Duke University Comprehesive Sickle Cell Center, Durham, N.C., 1989, pp. 53
Piomelli S, Lurinsky G, Masserrnan LR: The mechanism of red cell aging. 1. Relationship between cell age and specific gravity evaluated by ultracentrifugation in a discontinuous density gradient. J Lab Clin Med 69: 659–674, 1967
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Das, S.K., Mukherjee, S. Heterogeneity of human red blood cell membrane: Co-existence of heavy and light membranes. Mol Cell Biochem 196, 141–149 (1999). https://doi.org/10.1023/A:1006932010565
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DOI: https://doi.org/10.1023/A:1006932010565