Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer.
The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric and/or tetrameric band 3, and does not require postulation of a fraction of large clusters. Proteolytic removal of the cytoplasmic domain of band 3, which significantly increases the rotational mobility of the transmembrane domain, does not affect its oligomeric state, as reported by eosin homotransfer. These results support a model in which interaction with the membrane skeleton restricts the mobility of band 3 without significantly altering its self-association state.
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615 USA.
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D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008239:Lysine; D046911:Macromolecular Substances; D008555:Melitten / Q000494:pharmacology; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D015032:Zinc / Q000494:pharmacology
D001457:Anion Exchange Protein 1, Erythrocyte; D046911:Macromolecular Substances; D008555:Melitten; C033531:eosin maleimide; D015032:Zinc; D008239:Lysine; D004801:Eosine Yellowish-(YS)