Seed B, Sheen J Y
Department of Molecular Biology, Massachusetts General Hospital, Boston 02114.
Gene. 1988 Jul 30;67(2):271-7. doi: 10.1016/0378-1119(88)90403-9.
A simple and convenient phase extraction assay for chloramphenicol (Cm) acetyltransferase (CAT) activity has been developed, based on the enzymatic butyrylation of radiolabelled Cm. The assay is linear over two to three orders of magnitude of enzyme concentration, is highly sensitive, and substantially less expensive than all presently available alternatives. Methods for convenient CAT assay, adapted for mammalian cells, plant protoplasts, and mammalian cell culture supernatants, are described. These methods should also simplify measurement of CAT activity in other organisms, such as yeast and bacteria. In addition, a simple pre-extraction procedure is presented for purifying radiolabelled Cm which allows a 25-fold increase in sensitivity using tritiated substrates.
基于放射性标记氯霉素（Cm）的酶促丁酰化反应，开发了一种简单便捷的氯霉素乙酰转移酶（CAT）活性相萃取测定法。该测定法在酶浓度的两到三个数量级范围内呈线性，灵敏度高，且比目前所有可用的替代方法成本低得多。文中描述了适用于哺乳动物细胞、植物原生质体和哺乳动物细胞培养上清液的便捷CAT测定方法。这些方法也应能简化对其他生物体（如酵母和细菌）中CAT活性的测量。此外，还介绍了一种简单的预萃取程序，用于纯化放射性标记的Cm，使用氚标记底物时可使灵敏度提高25倍。
