Enzymatic methylation of band 3 anion transporter in intact human erythrocytes.
Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.
D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008745:Methylation; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000097:blood*; D014316:Tritium
D001457:Anion Exchange Protein 1, Erythrocyte; D014316:Tritium; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase