Magnesium-dependent tyrosine phosphatases

镁依赖性酪氨酸磷酸酶

• 批准号: 6109157
• 负责人: Jeremy Selengut
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6109157
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; aging; animal tissue; antibody formation; carbonate dehydratase; crosslink; enzyme activity; enzyme substrate; epitope mapping; immunoglobulin isotypes; immunoprecipitation; isozymes; laboratory rabbit; laboratory rat; liver; oxidative stress; protein purification; protein tyrosine phosphatase; recombinant proteins; western blottings

Carbonic anhydrase III (CA3) has been previously reported to have acid phosphatase activity, an observation made by several laboratories (including our own) studying CA3 purified from various species and tissues. Our present investigations of CA3 purified from rat liver and rabbit muscle, as well as the rat enzyme expressed in E. coli., however, do not support an intrinsic phosphatase activity for this enzyme. Rat CA3 was over-expressed in E. coli and purified by ammonium sulfate precipitation and DEAE, Cibachrome Blue, and phenyl-HIC chromatography. Phosphatase activity did not copurify with the recombinant CA3 in the final chromatography step. Also, phosphatase activity in the peak of wild-type CA3 protein falls well short of the reported values for the rabbit and pig muscle protein and slightly higher than that reported for bovine muscle. However, the majority of this activity is likely due to contamination from other, incompletely separated phosphatase peaks. We concluded from these studies that either, (a) expression in E. coli failed to confer an essential post-translational modification essential for phosphatase activity, (b) the active enzyme is a variant with slightly different elution on phenyl-HIC chromatography, (c) the active phosphatase requires a cofactor, which is removed by phenyl-HIC chromatography, or (d) the rat liver enzyme has an intrinsic activity which is even lower than that reported for the bovine muscle protein and was undetected by our methods. To distinguish among these possibilities, we returned to study enzyme purified directly from rat liver. Residual phosphatase activity in rat liver CA3 purified by the above procedure did not co-elute with CA3 on gel filtration. Also, antibodies raised to purified rat liver CA3 are able to precipitate CA3 from the phenyl-HIC-purified preparation but not the phosphatase activity. CA3 was then purified from rabbit muscle using essentially the same procedure as reported in the original studies on the phosphatase activity, and with similar results to those studies. Further purification of this material by high-resolution gel filtration, however, separates at least three contaminating phosphatases with apparent molecular weights of 19, 35, and 40 kDa. Residual phosphatase activity in the peak of CA3 protein was undetectable and far below the values reported in the literature. Care was taken in these studies to account for all of the phosphatase activity--no significant amount was lost suggesting that gel filtration did not remove an essential cofactor from CA3. These results indicate that CA3 has no intrinsic phosphatase activity.

碳酸酐酶III（CA 3）以前已被 据报道有酸性磷酸酶活性， 几个实验室（包括我们自己的）研究CA 3纯化 不同的物种和组织。我们目前对CA 3的研究 从大鼠肝脏和兔肌肉中纯化，以及大鼠酶 在大肠杆菌中表达大肠杆菌，但是，不支持内部 磷酸酶活性。大鼠CA 3过表达 在大肠大肠杆菌中，并通过硫酸铵沉淀进行纯化， DEAE、Cibachrome Blue和苯基-HIC色谱法。 磷酸酶活性不与重组CA 3共纯化， 最后的层析步骤。此外， 野生型CA 3蛋白的峰值福尔斯远低于报道的 兔和猪肌肉蛋白的值略高于 牛肌肉的报告。然而，其中大部分 活动可能是由于其他污染，不完全 分离的磷酸酶峰。我们从这些研究中得出结论， （a）在E.大肠杆菌未能赋予一个基本的 磷酸酶活性所必需的翻译后修饰， (b)活性酶是一种变体，其洗脱方式略有不同， 苯基-HIC色谱，（c）活性磷酸酶需要 辅因子，其通过苯基-HIC色谱法除去，或（d） 大鼠肝酶具有甚至更低内在活性 比牛肌肉蛋白报道的要高， 而不被我们的方法发现为了区分这些 可能性，我们回到研究酶纯化直接从大鼠 肝脏纯化的大鼠肝CA 3中的残余磷酸酶活性 上述程序在凝胶过滤上不与CA 3共沉淀。还有， 针对纯化的大鼠肝CA 3产生的抗体能够沉淀 CA 3来自苯基-HIC纯化的制备物，而不是 磷酸酶活性然后从兔肌肉中纯化CA 3， 使用与原始报告基本相同的程序， 磷酸酶活性的研究，并与那些类似的结果 问题研究通过高分辨率凝胶进一步纯化该材料 然而，过滤分离至少三种污染物， 表观分子量为19、35和40的磷酸酶 kDa。CA 3蛋白峰中残留的磷酸酶活性为 无法检测到并且远低于文献中报道的值。 在这些研究中注意考虑到所有的 磷酸酶活性--没有显著的损失，这表明， 凝胶过滤没有从CA 3中除去必需的辅因子。这些 结果表明，CA 3没有内在的磷酸酶活性。