Else A J, Barnes S J, Danson M J, Weitzman P D
Department of Biochemistry, University of Bath, U.K.
Biochem J. 1988 May 1;251(3):803-7. doi: 10.1042/bj2510803.
We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used this new assay in part of our investigations into the inhibitory effects of palmitoyl thioesters on diverse citrate synthases. Both palmitoyl-CoA and palmitoyl thioglycollate inhibit citrate synthases from pig heart, Bacillus megaterium and Escherichia coli, the E. coli enzyme showing the greatest sensitivity to these effectors. With palmitoyl-CoA the extent of inhibition is time-dependent, but the enzymes can be protected from the effect by the substrates oxaloacetate and acetyl-CoA. Using the dinitrophenylhydrazine assay, we have shown that the thioester bond is essential for inhibition; that is, if the palmitoyl thioesters are cleaved to give a mixture of palmitate and a thiol compound, the inhibitions of pig heart and B. megaterium citrate synthases are eliminated and that of the E. coli enzyme is markedly decreased.
我们已经证明，柠檬酸合酶可以通过一种基于用2,4-二硝基苯肼测定草酰乙酸利用情况的简单、间断分光光度法进行测定。该测定法适用于纯化的酶和细胞提取物，其优点是可以在高浓度硫醇和硫酯存在的情况下使用。我们已经在部分研究中使用这种新的测定法来研究棕榈酰硫酯对多种柠檬酸合酶的抑制作用。棕榈酰辅酶A和棕榈酰巯基乙酸盐均抑制猪心、巨大芽孢杆菌和大肠杆菌中的柠檬酸合酶，其中大肠杆菌的酶对这些效应物最为敏感。对于棕榈酰辅酶A，抑制程度与时间有关，但草酰乙酸和乙酰辅酶A等底物可以保护酶免受这种影响。使用二硝基苯肼测定法，我们已经表明硫酯键对于抑制作用至关重要；也就是说，如果棕榈酰硫酯被裂解生成棕榈酸和硫醇化合物的混合物，猪心和巨大芽孢杆菌柠檬酸合酶的抑制作用就会消除，而大肠杆菌酶的抑制作用则会显著降低。
