Reversible methylation of cytoskeletal and membrane proteins in intact human erythrocytes.
Membrane protein methylation was studied in intact human erythrocytes. Erythrocytes were incubated with physiological concentrations of L-[methyl]3H]methionine and incorporated 35 pmol of methyl groups into membrane components/mg of membrane protein in a 2.5-hr incubation at 37 degrees C. At least 90% of the total groups (12,500 methyl groups/cell) were incorporated into polypeptides via linkages which were labile to 1 N NaOH. Major methylated membrane polypeptides were identified based on the comigration of radioactivity with Coomassie blue- and periodic acid-Schiff-staining species in pH 2.4 dodecyl sulfate gel electrophoresis, as well as by the distribution of radiolabel and protein following selective proteolysis and membrane extraction procedures. Methylated species identified in this way include the cytoskeletal polypeptides band 2.1 (ankyrin) and band 4.1, as well as the band 3 anion transport protein. Other methylated species include an intrinsic polypeptide comigrating with band 3 but insensitive to external chymotrypsin digestion, a glycoprotein showing variable migration in this gel system (40,000-55,000 daltons), an intrinsic polypeptide at about 30,000 daltons, and an extrinsic species of about 17,000 daltons. A small amount of radioactivity comigrated with the band 4.5 region. Bands 1, 2, 4.2, 5, 6, 7, and the major sialoglycoprotein were not methylated in this system. All of the methylated species exhibited turnover in vivo, and the time taken to reach 50% demethylation for each species ranged from less than 2 to 29 h.
D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000097:blood*; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D014357:Trypsin
D008565:Membrane Proteins; D010455:Peptides; D008715:Methionine; D002918:Chymotrypsin; D014357:Trypsin