Antonsson B, Barredo J, Moran R G
Department of Biochemistry, University of Southern California, Los Angeles.
Anal Biochem. 1990 Apr;186(1):8-13. doi: 10.1016/0003-2697(90)90563-o.
A new assay for the enzyme folylpoly-gamma-glutamate synthetase (FPGS) that offers significant advantages over other published procedures has been developed. This assay is based on the addition of high specific activity [3H]glutamic acid to (6-S)-tetrahydrofolate followed by trapping of the labeled tetrahydropteroyldiglutamate product as a covalently bound macromolecular complex by the addition of formaldehyde, fluorodeoxyuridylate, and pure bacterial thymidylate synthase. This complex is then separated from excess labeled glutamic acid by centrifugal elution of a 1-ml Sephadex G-50 column. The assay was found to be useful for the measurement of FPGS on small tissue samples and is amenable with the assay of FPGS in cell sonicates. Typically, blank values of 100-200 cpm are seen with a signal normally more than 10 times higher. Analysis of 20-30 samples can be accomplished in less than 90 min. As a result, this assay has proven useful for detection of enzyme in elution fractions from chromatographic columns.
已开发出一种针对叶酸多聚 -γ- 谷氨酸合成酶（FPGS）的新检测方法，该方法相较于其他已发表的方法具有显著优势。此检测方法基于向（6 - S）- 四氢叶酸中添加高比活度的[³H]谷氨酸，随后通过添加甲醛、氟脱氧尿苷酸和纯细菌胸苷酸合成酶，将标记的四氢蝶酰二谷氨酸产物捕获为共价结合的大分子复合物。然后通过1毫升葡聚糖凝胶G - 50柱的离心洗脱，将该复合物与过量的标记谷氨酸分离。该检测方法被发现可用于测量小组织样本中的FPGS，并且适用于细胞超声裂解液中FPGS的检测。通常，空白值为100 - 200 cpm，而信号通常比空白值高10倍以上。在不到90分钟的时间内可以完成20 - 30个样本的分析。因此，该检测方法已被证明可用于检测色谱柱洗脱级分中的酶。
