Barber J R, Clarke S
J Biol Chem. 1983 Jan 25;258(2):1189-96.
The level of carboxyl methylation of membrane proteins has been measured in intact human erythrocyte populations of different ages separated by density gradient centrifugation. Age separation was confirmed by measurement of cytosolic pyruvate kinase specific activity in each fraction. When cells of different ages were incubated with L-[methyl-3H]methionine, the steady state level of 3H radioactivity covalently bound to membrane proteins is observed to be at least 3-fold higher in older erythrocytes. Because the specific radioactivity of the methyl group donor S-adenosyl-L-[methyl-3H]methionine was identical in all age fractions, this represents an increase in the extent of modification of membrane proteins by carboxyl methylation. Of the three major methylated erythrocyte membrane proteins, this increase in carboxyl methylation with age is 4 to 7-fold for bands 2.1 and 3, while the increase in band 4.1 is 3 to 4-fold. This increase in the steady state level of methylation with age cannot be explained by changes in either the intrinsic rate of methyl transfer or by changes in the rate constant of methyl turnover. We, therefore, propose that the age-dependent change in carboxyl methylation is due to an increase in the number of available acceptor sites as the erythrocyte ages in vivo. Since methylation of acidic residues on erythrocyte membrane proteins has been detected exclusively on D-aspartic acid residues (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464), these results are consistent with an accumulation of D-aspartic acid in membrane protein due to spontaneous racemization a the cell ages. The relationship of these observations to possible functions of erythrocyte membrane protein carboxyl methylation is discussed.
通过密度梯度离心分离出不同年龄的完整人类红细胞群体，测量了膜蛋白的羧基甲基化水平。通过测量每个组分中胞质丙酮酸激酶的比活性来确认年龄分离情况。当不同年龄的细胞与L-[甲基-³H]甲硫氨酸一起孵育时，观察到与膜蛋白共价结合的³H放射性的稳态水平在衰老红细胞中至少高3倍。由于甲基供体S-腺苷-L-[甲基-³H]甲硫氨酸的比放射性在所有年龄组分中相同，这代表了膜蛋白羧基甲基化修饰程度的增加。在三种主要的甲基化红细胞膜蛋白中，随着年龄增长，带2.1和带3的羧基甲基化增加了4至7倍，而带4.1的增加为3至4倍。甲基化稳态水平随年龄的这种增加，既不能用甲基转移的内在速率变化来解释，也不能用甲基周转的速率常数变化来解释。因此，我们提出，羧基甲基化随年龄的变化是由于红细胞在体内衰老时可利用的受体位点数量增加所致。由于红细胞膜蛋白酸性残基的甲基化仅在D-天冬氨酸残基上被检测到（麦克法登，P. N.，和克拉克，S.（1982年）《美国国家科学院院刊》79，2460 - 2464），这些结果与随着细胞衰老，膜蛋白中D-天冬氨酸因自发消旋而积累一致。讨论了这些观察结果与红细胞膜蛋白羧基甲基化可能功能的关系。
