Crabb D W, Dixon J E
Anal Biochem. 1987 May 15;163(1):88-92. doi: 10.1016/0003-2697(87)90096-0.
Transfection of several cell lines (HeLa, COS, PC-12, CA-77, and H4IIE C3) with pRSV-CAT by a variety of methods yielded rather low chloramphenicol acetyltransferase (CAT) activity in cell extracts. Extracts of these cells were found to interfere with the assay of added CAT. The extracts were capable of deacetylating acetylchloramphenicol and of accelerating the rate of hydrolysis of the acetyl-CoA present in the assay. Heating the cell extract to 60 degrees C for 10 min completely prevented the interference and slowed the hydrolysis of acetyl-CoA. Substantially higher CAT activities were observed when the extract was heat treated in the presence of EDTA prior to enzyme assay for most cell lines tested. This simple reliable method makes possible the accurate assessment of CAT activities in different cell lines. These observations are particularly pertinent to investigators studying tissue-specific gene expression.
通过多种方法用pRSV - CAT转染几种细胞系（HeLa、COS、PC - 12、CA - 77和H4IIE C3），在细胞提取物中产生的氯霉素乙酰转移酶（CAT）活性相当低。发现这些细胞的提取物会干扰添加的CAT的测定。提取物能够使乙酰氯霉素脱乙酰化，并加速测定中存在的乙酰辅酶A的水解速率。将细胞提取物加热至60摄氏度10分钟可完全消除干扰并减缓乙酰辅酶A的水解。对于大多数测试的细胞系，在酶测定之前，当提取物在EDTA存在下进行热处理时，观察到CAT活性显著更高。这种简单可靠的方法使得准确评估不同细胞系中的CAT活性成为可能。这些观察结果对于研究组织特异性基因表达的研究人员尤为相关。
