title: Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes.
abstract: Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50 degrees C for 15 min. The membrane skeleton dissociation/association was determined by measuring the Tris-induced dissociation of Triton-insoluble membrane skeletons (Triton shells), the spectrin-actin extractability under low ionic conditions, and the binding of spectrin-actin with normal erythrocyte membrane inside-out vesicles (IOVs). The spectrin structure was ascertained by measuring the spectrin dimer-to-tetramer ratio as well as the spectrin tryptophan fluorescence. Both the Tris-induced Triton shell dissociation and the spectrin-actin extractability under low ionic conditions were considerably reduced by the heat treatment. Also, the binding of heated erythrocyte spectrin-actin to IOVs was significantly smaller than that observed with the normal cell spectrin-actin. Further, the quantity of spectrin dimers was appreciably increased in heat-treated erythrocytes as compared to the normal cells. This change in the spectrin dimer-to-tetramer ratio was accompanied by marked changes in the spectrin tryptophan fluorescence. In spite of these heat-induced alterations in structure and bilayer interactions of the membrane skeleton, the inside-outside glycerophospholipid distribution remained virtually unaffected in the heat-treated cells, as judged by employing bee venom and pancreatic phospholipase A2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skeleton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane.
authors.affiliation: Division of Membrane Biology, Central Drug Research Institute, Lucknow, India.
authors.forename: S R
authors.initials: SR
authors.lastname: Gudi
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: V
authors.initials: V
authors.lastname: Bhakuni
authors.forename: S M
authors.initials: SM
authors.lastname: Gokhale
authors.forename: C M
authors.initials: CM
authors.lastname: Gupta
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000097:blood / Q000378:metabolism; D003599:Cytoskeleton / Q000201:enzymology / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D004912:Erythrocytes / Q000378:metabolism*; D005450:Fluorescamine; D005978:Glutathione / Q000097:blood / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000097:blood / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism
chemical_list: D000199:Actins; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010743:Phospholipids; D013049:Spectrin; D005450:Fluorescamine; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D005978:Glutathione