Increased methyl esterification of membrane proteins in aged red‐blood cells

Abstract

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins was investigated in 3 different age-related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37.degree. C) in the presence of L-[methyl-3H]methionine, the precursor in vivo of the methyl donor S-adenosylmethionine, a 4-fold increase in membrane-protein carboxyl methylation was observed in the oldest cells compared with the youngest ones. The identification of methylated species shows in all cell fractions a pattern similar to that reported for unfractionated erythrocytes; this is based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell aging. Particularly in band 4.1 the age-related increase in methyl esterification is accompanied by a significant reduction of the half-life of methyl esters. The activity of cytoplasmic protein methylase II does not change during cell aging, while the isolated ghosts from erythrocytes of different age show an age-related increased ability to act as methyl-accepting substrates, when incubated in presence of purified protein methylase II and methyl-labeled S-adenosylmethionine; therefore, the relevance of membrane structure in determining membrane protein methylation levels can be postulated. Finally the possible correlation of this post-translational protein modification with erythrocyte aging is discussed.