A combination of vectorial modification and site-directed immunochemistry has been used to determine the disposition, with respect to the membrane, of Lys-691 of the anion exchanger from human erythrocytes. Intact erythrocytes and inside-out vesicles were vectorially modified in the same container with pyridoxal phosphate and sodium [3H]borohydride. The modified inside-out vesicles were separated from erythrocytes by differential centrifugation and the vesicles and erythrocyte membranes were treated with alkali and digested with trypsin and thermolysin to liberate the peptides IVSKPER and IVSK{Nε-[4´-(5´-phospho-[4-3H]pyridoxyl)]}PER. These peptides, containing the unmodified and modified versions of Lys-691, were retrieved from the digests by site-directed immunochemistry and were identified by HPLC and liquid scintillation spectroscopy. Both the inside-out vesicles and the intact erythrocytes contained the peptide IVSKPER, however, the 3H-label from the phosphopyridoxylated peptide could be detected only in the inside-out vesicles. The incorporation of 3H into Lys-691 of the anion exchanger from inside-out vesicles was at least 30-fold greater than the incorporation into Lys-691 of the anion exchanger from intact erythrocytes. It follows that Lys-691 of the anion exchanger is located on the cytoplasmic surface of the plasma membrane.

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