Biochimica et Biophysica Acta, 1023 (1990) 63-72 63 Elsevier BBAMEM 74767 Membrane skeleton-bilayer interaction is not the major determinant of membrane phospholipid asymmetry in human erythrocytes * S.R.P. Gudi, A. Kumar * *, V. Bhakuni, S.M. Gokhale and C.M. Gupta Division of Membrane Biology, Central Drug Research Institute, Lucknow (India) (Received 23 October 1989) Key words: Phospholipid asymmetry; Membrane skeleton; Spectrin denaturation; (Human) Transbilayer phospholipid distribution, membrane skeleton dissociation/association, and spectrin structure have been analysed in human erythrocytes after subjecting them to heating at 50°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 beat-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. Inspite of these beat-induced alterations in structure and hilayer 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 A 2, fluorescamine and Merocyanine 540 as the external membrane probes. These results strongly indicate that membrane bilayer-skele- ton interaction is not the major factor in determining the transbilayer phospholipid asymmetry in human erythrocyte membrane. Introduction Human erythrocyte membrane is composite of two structural units, viz. membrane skeleton (or cyto- skeleton) and membrane bilayer. While the membrane bilayer is comprised of lipids and integral membrane * Communication No. 4063 from C.D.R.I., Lucknow, India. * * Present address: Department of Pathology,Yale University, School of Medicine, 310 Cedar Street, New Haven, CT 06510, U.S.A. Abbreviations: RBC, red blood cells; PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; PS, phosphatidyl- serine; PMSF, phenylmethylsulfonyl fluoride; GSH, glutathione; ATP, adenosine 5'-triphosphate; EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecylsulfate; PBS, phosphate-bufferedsaline; Mc 540, Merocyanine 540; IOVs, inside-out vesicles; ROVs, fight side-out vesicles; TLC, thin-layer chromatography. Correspondence: C.M. Gupta, Division of Membrane Biology, Central Drug Research Institute, Lucknow 226 001, India. proteins, membrane skeleton is a reticulate type of structure formed from three major (spectrin, actin and polypeptide 4.1) and several minor peripheral mem- brane proteins and is associated with the cytoplasmic face of the membrane bilayer through protein-protein and protein-phospholipid interactions [1]. The mem- brane bilayer-skeleton association controls not only the membrane mechanical stability and deformability but also the lateral mobility of the integral membrane pro- teins [2]. Besides, this association has been considered as the major factor in maintaining the asymmetric phos- pholipid distribution across the erythrocyte membrane bilayer [3]. Erythrocyte membrane phospholipids are asymmetri- cally distributed in the two halves of the membrane bilayer. The choline-containing phospholipids (PC and SM) are present mainly in the outer monolayer, whereas the aminophospholipids (PE and PS) are localized al- most exclusively in the inner monolayer [4]. This typical transbilayer phospholipid asymmetry is disturbed in 0005-2736/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)