Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.
Amplification and detection of substrates for protein carboxyl methyltransferases in PC12 cells.
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PC12 细胞中蛋白质羧甲基转移酶底物的扩增和检测。
DOI：
10.1016/0003-2697(91)90413-n
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发表时间：
1991
影响因子：
2.9
通讯作者：
Aswad,DW
中科院分区：
文献类型：
--
作者：
Najbauer,J;Johnson,BA;Aswad,DW
一种有助于鉴定形成“稳定”甲酯的蛋白质羧基甲基转移酶的底物的策略，即，描述了在常规聚丙烯酰胺凝胶电泳过程中基本上保持完整的那些。大鼠PC 12细胞在腺苷二醛（甲基化抑制剂）的存在下培养，以促进低甲基化蛋白的积累。然后在S-[甲基-3H]腺苷-1-甲硫氨酸存在下孵育Nonidet P-40细胞提取物，以通过内源性甲基转移酶标记甲基接受位点。标记蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离后，凝胶切片在4 n甲磺酸或6 n盐酸中孵育以水解甲酯。通过在液体闪烁液中捕获检测所得[3 H]甲醇。观察到7个羧基甲基化蛋白质，质量范围为18至96 kDa。检测这些蛋白质中的五种需要用腺苷二醛预先处理细胞，而甲基掺入到18 kDa的一种蛋白质中的处理大大增强。使用酸性条件进行甲酯水解相对于使用碱性水解条件的测定具有重要优势。在PC 12细胞中，以及可能存在显著水平精氨酸甲基化的其他细胞类型中，甲醇信号被碱性条件下产生的高水平挥发性甲胺所掩盖。在酸性条件下进行扩散测定可消除这种干扰。腺苷二醛，凭借增加的甲基接受能力的底物蛋白质羧基甲基转移酶，结合更有选择性的测定羧基甲基化，应证明有用的分离和表征新的蛋白质羧基甲基转移酶及其底物。
A strategy that facilitates the identification of substrates for protein carboxyl methyltransferases that form “stable” methyl esters, i.e., those that remain largely intact during conventional polyacrylamide gel electrophoresis is described. Rat PC12 cells were cultured in the presence of adenosine dialdehyde (a methylation inhibitor) to promote the accumulation of hypomethylated proteins. Nonidet P-40 cell extracts were then incubated in the presence of S-[methyl-3H]adenosyl-l-methionine to label methyl-accepting sites via endogenous methyltransferases. After labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel slices were incubated in 4 n methanesulfonic acid or 6 n HCl to hydrolyze methyl esters. The resulting [3H]methanol was detected by trapping in liquid scintillation fluid. Seven carboxyl methylated proteins were observed with masses ranging from 18 to 96 kDa. Detection of five of these proteins required prior treatment of cells with adenosine dialdehyde, while methyl incorporation into one protein at 18 kDa was substantially enhanced by the treatment. The use of acidic conditions for methyl ester hydrolysis has an important advantage over assays that utilize alkaline hydrolysis conditions. In PC12 cells, and possibly other cell types where there are significant levels of arginine methylation, the methanol signal becomes obscured by high levels of volatile methylamines generated under the alkaline conditions. Carrying out diffusion assays under acidic conditions eliminates this interferance. Adenosine dialdehyde, by virtue of increasing the methyl-accepting capacity of substrates for protein carboxyl methyltransferases, in combination with a more selective assay for carboxyl methylation, should prove useful in the isolation and characterization of new protein carboxyl methyltransferases and their substrates.