Cheek W D, Appling D R
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712.
Arch Biochem Biophys. 1989 May 1;270(2):504-12. doi: 10.1016/0003-9861(89)90532-8.
C1-tetrahydrofolate synthase (C1-THF synthase), a eukaryotic trifunctional enzyme, catalyzes three sequential folate-mediated one-carbon interconversions. These three reactions supply the activated one-carbon units required in the metabolism of purines, thymidylate, and several amino acids. In order to study the regulation of C1-THF synthase expression in mammals, we have purified the enzyme to homogeneity from rat liver, raised polyclonal antisera to it in rabbits, and developed a sensitive solid-phase immunoassay for the enzyme. The enzyme was purified approximately 600-fold to a specific activity of 24.6 U/mg protein based on 10-formyl-THF synthetase activity. Western blot analysis indicated that the antisera is specific for one protein in crude liver extracts which comigrates with purified C1-THF synthase. Using the solid-phase immunoassay, as little as 200 pg of immunoreacting protein can be detected in tissue homogenates. Several rat tissues were examined for the three C1-THF synthase enzymatic activities and immunoreactive protein. The results indicated that the level of C1-THF synthase is regulated in a tissue-specific manner. Enzyme assays revealed that certain tissues differ by more than 100-fold in enzyme activity, with liver and kidney containing the highest levels, and lung and muscle the lowest. However, immunoassay of these same tissues indicated only a 10-fold difference in C1-THF synthase concentration. This apparent masking of enzyme activity was observed in all tissues, but to varying degrees. These results emphasize the advantages of an immunoassay in studying the regulation of C1-THF synthase.
C1-四氢叶酸合酶（C1-THF合酶）是一种真核生物三功能酶，催化三个连续的叶酸介导的一碳单位相互转化反应。这三个反应提供了嘌呤、胸苷酸和几种氨基酸代谢所需的活化一碳单位。为了研究哺乳动物中C1-THF合酶表达的调控，我们已从大鼠肝脏中将该酶纯化至同质，在兔中制备了针对它的多克隆抗血清，并开发了一种针对该酶的灵敏固相免疫测定法。基于10-甲酰-THF合成酶活性，该酶被纯化了约600倍，比活性达到24.6 U/mg蛋白质。蛋白质印迹分析表明，该抗血清对粗肝提取物中的一种蛋白质具有特异性，该蛋白质与纯化的C1-THF合酶迁移率相同。使用固相免疫测定法，在组织匀浆中可检测到低至200 pg的免疫反应性蛋白。检测了几种大鼠组织中的三种C1-THF合酶酶活性和免疫反应性蛋白。结果表明，C1-THF合酶的水平以组织特异性方式受到调控。酶活性测定显示，某些组织的酶活性相差100倍以上，肝脏和肾脏中的水平最高，而肺和肌肉中的水平最低。然而，对这些相同组织的免疫测定表明，C1-THF合酶浓度仅相差10倍。在所有组织中均观察到这种酶活性的明显掩盖，但程度不同。这些结果强调了免疫测定法在研究C1-THF合酶调控方面的优势。
