Crabb D W, Minth C D, Dixon J E
Methods Enzymol. 1989;168:690-701. doi: 10.1016/0076-6879(89)68050-0.
These experiments document the presence of enzymatic activities in extracts of commonly used cell lines which interfere with the determination of CAT activity. We suspect that the deacetylase activity is the most important, as the extract of the H4IIE C3 cells was capable of completely deacetylating the mono- and diacetylchloramphenicol formed during a 2-hr incubation of CAT with chloramphenicol and acetyl-CoA. The results of the inhibitor experiments are consistent with the presence of proteases which degrade CAT, or a serine carboxylesterase. The interference was also reduced by about half by EDTA; a metalloenzyme (either a protease or esterase) may therefore be involved. This interference appears to be a common phenomenon. We have surveyed 23 different cell types for the presence of the interfering activity and found it in 15. The interference was particularly prominent in several neuroendocrine and hepatoma cells. We took advantage of the effect of EDTA and the heat stability of CAT to eliminate the interference. Addition of 5 mM EDTA and a 10-min incubation of the sonicated cell suspension at 60 degrees prior to centrifugation abolished the interference in all cell lines tested. It is important to note that in order to reveal any CAT activity in some of the extracts (e.g., PC-12 or Hep3B), it was necessary to run the CAT assay for 2 hr. The control assays were therefore run almost to completion, and were well beyond the linear range of the assay. Therefore, the small differences which we observed between the heat-treated and control samples in some instances (e.g., rice, corn, or HeLa cells) will be dramatically amplified when the CAT assay is performed under conditions in which only a small percentage of the substrate is converted to product. After these studies had been performed, we found that others have also recommended heat treatment of the cell extract prior to CAT assay. We concur with this recommendation. We suggest that EDTA plus heat treatment of the cell extract should be incorporated into all CAT assay protocols, unless it has been previously determined that extracts of the cells used do not interfere. Furthermore, the heat treatment step should be used whenever the activity of promoter-CAT constructs is compared among different cell lines, as is often done to define tissue-specific expression.
这些实验证明了常用细胞系提取物中存在干扰氯霉素乙酰转移酶（CAT）活性测定的酶活性。我们怀疑脱乙酰酶活性最为重要，因为H4IIE C3细胞的提取物能够使CAT与氯霉素和乙酰辅酶A在2小时孵育过程中形成的单乙酰氯霉素和二乙酰氯霉素完全脱乙酰化。抑制剂实验结果与存在降解CAT的蛋白酶或丝氨酸羧酸酯酶一致。EDTA也使干扰减少了约一半；因此可能涉及一种金属酶（蛋白酶或酯酶）。这种干扰似乎是一种常见现象。我们调查了23种不同细胞类型中是否存在干扰活性，发现其中15种存在该活性。干扰在几种神经内分泌细胞和肝癌细胞中尤为突出。我们利用EDTA的作用和CAT的热稳定性来消除干扰。在离心前，向超声处理的细胞悬液中加入5 mM EDTA并在60℃孵育10分钟，消除了所有测试细胞系中的干扰。需要注意的是，为了在某些提取物（如PC - 12或Hep3B）中检测到任何CAT活性，有必要进行2小时的CAT测定。因此对照测定几乎进行到完成，且超出了测定的线性范围。所以，当在只有一小部分底物转化为产物的条件下进行CAT测定时，我们在某些情况下（如水稻、玉米或HeLa细胞）观察到的热处理样品与对照样品之间的微小差异会被显著放大。在进行这些研究之后，我们发现其他人也建议在进行CAT测定之前对细胞提取物进行热处理。我们赞同这一建议。我们建议，除非之前已确定所用细胞的提取物无干扰，否则应将EDTA加细胞提取物热处理纳入所有CAT测定方案中。此外，每当在不同细胞系之间比较启动子 - CAT构建体的活性时（这在定义组织特异性表达时经常进行），都应使用热处理步骤。
