O'Connor C M, Germain B J
J Biol Chem. 1987 Jul 25;262(21):10404-11.
Transmethylation reactions in fully grown Xenopus oocytes were analyzed following the microinjection of S-adenosyl-L-[methyl-3H]methionine (AdoMet). The size of the endogenous AdoMet pool, measured by cation exchange high pressure liquid chromatography is 5.91 pmol/oocyte. The AdoMet pool turns over with a half-time of 2 h, at a rate of 2.07 pmol/h/oocyte. Fractionation experiments indicate that approximately one-third of the AdoMet in oocytes is utilized for protein carboxylmethylation reactions and another third is metabolized into small molecules which are secreted. The remainder of the intracellular AdoMet is used primarily for protein N-methylation reactions, although some methylation of phospholipids and nucleic acids also occurs. Polyacrylamide gel electrophoresis of 3H-methylated proteins at pH 2.4 in the presence of sodium dodecyl sulfate demonstrated that methyl esters are associated with a heterogeneous group of proteins in both the nucleus and cytoplasm of oocytes, coincident with the subcellular distribution of the protein D-aspartyl, L-isoaspartyl methyl transferase (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). The protein methyl esters associated with oocyte proteins turn over rapidly, as evidenced from the presence of [3H]methanol in the medium. The calculated rate of protein carboxyl methylation, 0.7 pmol/h/oocyte, is similar to that of protein synthesis in oocytes, suggesting that the modification of derivatized aspartyl residues represents a major pathway in oocyte protein metabolism. Since the formation of protein methyl esters is unaffected by cycloheximide, it is unlikely that methyl-accepting sites on oocyte proteins arise primarily from errors in protein synthesis. Unlike protein carboxyl methylation reactions, protein N-methylation reactions are closely linked to protein synthesis, and the methyl group linkages are stable over a period of at least 4 h. Numerous protein acceptors for N-methylation reactions were identified by polyacrylamide gel electrophoresis.
在向完全成熟的非洲爪蟾卵母细胞显微注射S-腺苷-L-[甲基-³H]甲硫氨酸（AdoMet）后，对其中的转甲基化反应进行了分析。通过阳离子交换高压液相色谱法测得内源性AdoMet库的大小为5.91皮摩尔/卵母细胞。AdoMet库的周转半衰期为2小时，速率为2.07皮摩尔/小时/卵母细胞。分级分离实验表明，卵母细胞中约三分之一的AdoMet用于蛋白质羧甲基化反应，另外三分之一代谢为分泌的小分子。细胞内其余的AdoMet主要用于蛋白质N-甲基化反应，不过也会发生一些磷脂和核酸的甲基化。在十二烷基硫酸钠存在下，于pH 2.4条件下对³H-甲基化蛋白质进行聚丙烯酰胺凝胶电泳，结果表明甲酯与卵母细胞核和细胞质中的一组异质蛋白质相关联，这与蛋白质D-天冬氨酰-L-异天冬氨酰甲基转移酶的亚细胞分布一致（奥康纳，C.M.（1987年）《生物化学杂志》262卷，10398 - 10403页）。从培养基中存在[³H]甲醇可证明，与卵母细胞蛋白质相关的蛋白质甲酯周转迅速。计算得出的蛋白质羧甲基化速率为0.7皮摩尔/小时/卵母细胞，与卵母细胞中的蛋白质合成速率相似，这表明衍生化天冬氨酰残基的修饰是卵母细胞蛋白质代谢的主要途径。由于蛋白质甲酯的形成不受环己酰亚胺影响，所以卵母细胞蛋白质上的甲基接受位点不太可能主要源于蛋白质合成错误。与蛋白质羧甲基化反应不同，蛋白质N-甲基化反应与蛋白质合成紧密相关，并且甲基连接在至少4小时内是稳定的。通过聚丙烯酰胺凝胶电泳鉴定出了许多N-甲基化反应的蛋白质受体。
