Incorporation of L-azetidine-2-carboxylic acid into hemoglobin in rabbit reticulocytes in vitro

Abstract

L-Azetidine-2-carboxylic acid is the naturally occurring lower homologue of L-proline. Reticulocytes from anemic rabbits incubated with DL-[14-C]azetidine-2-carboxylic acid synthesized radiolabeled hemoglobin, which when isolated from cell lysates co-chromatographed with unlabeled hemoglobin on Sephadex G-100 columns. Amino acid analysis of hemoglobin from reticulocytes incubated with DL-[14-C]-azetidine-2-carboxylic acid suggested that the homologue was incorporated into hemoglobin intact and unaltered. Alternatively, another amino acid analogue, 1-aminocyclopentane-[1-14-C]carboxylic acid, which is purported to be a valine antagonist, was not incorporated into hemoglobin under these conditions. Incubation of reticulocytes with 1, 5, and 10 mM L-azetidine-2-carboxylic acid reduced L-[U-14-C]proline (0.10 mM) incorporation into hemoglobin by 25, 58, and 72%, respectively. Conversely, 1.45 and 145 muM L-proline reduced radiolabeled azetidine-2-carboxylic acid (0.8 mM) in corporation into hemoglobin by 45 and 92%, respectively. Incorporation of L-[U-14-C]leucine and L-[U-14-C]lysine (0.1 mM each) into hemoglobin was unaffected at these concentrations of L-azetidine-2-carboxylic acid. These results suggest that L-azetidine-2-carboxylic acid is incorporated into hemoglobin without reducing the rate of globin synthesis in rabbit reticulocytes in vitro. The alpha and beta chains of hemoglobin into which [14-C]azetidine-2-carboxylic acid had been incorporated in rabbit reticulocytes in vitro were resolved electrophoretically on sodium dodecyl sulfate-polyacrylamide gels. The ratio of total radioactivity in the alpha and beta chains separately extracted from gels was in good agreement with the known 7:4 ratio of prolyl residues in the respective chains. Autoradiograms of two-dimensional tryptic peptide maps of rabbit globin into which either [14-C]azetidine-2-carboxylic acid or [14-C]proline had been incorporated showed nearly identical patterns of radioactivity. These results suggest that azetidine-2-carboxylic acid substitutes specifically for prolyl residues during in vitro hemoglobin synthesis in rabbit reticulocytes.