Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes.
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.
Gudi
S R
SR
Division of Membrane Biology, Central Drug Research Institute, Lucknow, India.
Kumar
A
A
Bhakuni
V
V
Gokhale
S M
SM
Gupta
C M
CM
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
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