A tightly membrane-associated subpopulation of spectrin is 3H-palmitoylated.
A tightly membrane-associated form of spectrin (TMA-spectrin) was labeled when human red blood cells were incubated with [3H]palmitic acid. About 90% of spectrin was not fatty acid-acylated and was extracted from membranes by low salt buffers. The 3H-palmitoylated TMA-spectrin, however, resisted low and even high salt extraction and remained associated with inside-out vesicles that were generated in the process of spectrin-actin extraction from membranes. TMA-spectrin was preferentially extracted from KCl-stripped vesicles by 5 M urea at low ionic strength. TMA-spectrin was purified by gel filtration and by ion exchange chromatography in the presence of urea and a non-ionic detergent. Purified TMA-spectrin was 3H-palmitoylated exclusively in the beta subunit to 0.28 mol/mol after a 12-h incubation of red cells. The labeled palmitate may be bound as an ester or thioester, since hydroxylamine (1 M, pH 7.5) released the label completely. Peptide maps of 3H-palmitoylated TMA-spectrin showed three or two labeled peptides from the beta subunit, when generated by V8 protease and trypsin, respectively. Two types of antibodies to spectrin reacted with purified TMA-spectrin, and TMA-spectrin contained the same antigenic peptides as low salt-extractable spectrin. Rabbit anti-ankyrin antibodies did not bind to TMA-spectrin. The substoichiometric incorporation of [3H]palmitic acid into TMA-spectrin could result from the slow turnover of endogenously bound fatty acids. Generation of the tightly membrane-associated and 3H-palmitoylated subpopulation of spectrin cannot be due to entrapment of an unmodified residual fraction of spectrin in right-side-out vesicles. Instead, the data suggest the existence of a subpopulation of spectrin molecules that undergo a covalent fatty acid modification and thereby alter their binding properties. This may offer a new, metabolically dependent mechanism for dynamic interactions between spectrin and the membrane lipid bilayer.
Laboratory for Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, Zurich.
M
M
Mariani
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D
Maretzki
H U
HU
Lutz
D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000648:ultrastructure; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000097:blood*; D013049:Spectrin / Q000302:isolation & purification / Q000378:metabolism*; D014316:Tritium; D014461:Ultracentrifugation; D014508:Urea
D008565:Membrane Proteins; D010169:Palmitic Acids; D014316:Tritium; D013049:Spectrin; D019308:Palmitic Acid; D014508:Urea