Prise K M, Gillies N E, Whelan A, Newton G L, Fahey R C, Michael B D
Cancer Research Campaign Gray Laboratory, Mount Vernon Hospital, Northwood, UK.
Int J Radiat Biol. 1995 Apr;67(4):393-401. doi: 10.1080/09553009514550451.
The role of net charge (zeta) of thiols in their ability to radioprotect cells has been investigated in a glutathione (GSH)-deficient strain of E. coli. This strain, 7, is deficient in the enzyme gamma-glutamylcysteine synthetase and allows the effects of added low molecular weight thiols to be studied. Using the gas explosion system it is possible to measure the chemical repair of the free-radical precursors of lethal lesions by thiols in intact cells. The first-order chemical repair rate in strain 7 is 280 s-1 in comparison with 1100 s-1 in the wild-type strain 1157. From the measured difference in the intracellular concentration of GSH between the wild-type and the mutant, this gives a second-order repair rate, kr, of 1.23 +/- 0.3 x 10(5) dm3mol-1s-1. Measurement of intracellular thiol levels after addition of various low molecular weight thiols showed that uptake was rapid, leading to stable thiol levels within 1 min. The ratios of the intracellular to extracellular concentrations (Cin/Cout) were 0.74 for 3-mercaptopropionic acid (zeta = -1), 0.56 for 2-mercaptoethanol (zeta = 0), 1.47 for cysteamine (zeta = +1) and 1.04 for WR1065 (zeta = +2). The kr's for these thiols were 1.3 +/- 0.5 x 10(5) dm3mol-1s-1 for 30-mercaptopropionic acid, 3.3 +/- 1.6 x 10(5) dm3mol-1s-1 for 2-mercaptoethanol, 3.9 +/- 1.1 x 10(5) dm3mol-1s-1 for cysteamine and 2.7 +/- 1.1 x 10(6) dm3mol-1s-1 for WR1065. These are lower and increase less with charge than previously published values for chemical repair in isolated pBR322 DNA, probably because of the association of nucleoproteins and polyamines with the cellular DNA of E. coli. However, the approximate three-fold increase in kr per unit increase in zeta shows that the counter-ion condensation and co-ion depletion are important in determining the effectiveness of charged thiols in the radioprotection of E. coli.
在一株缺乏谷胱甘肽（GSH）的大肠杆菌中，研究了硫醇的净电荷（ζ）在其对细胞辐射防护能力中的作用。该菌株7缺乏γ-谷氨酰半胱氨酸合成酶，可用于研究添加的低分子量硫醇的作用。使用气体爆炸系统，可以测量完整细胞中硫醇对致死性损伤自由基前体的化学修复作用。菌株7中的一级化学修复速率为280 s-1，而野生型菌株1157中的为1100 s-1。根据野生型和突变体之间GSH细胞内浓度的测量差异，得出二级修复速率kr为1.23±0.3×105 dm3mol-1s-1。添加各种低分子量硫醇后对细胞内硫醇水平的测量表明，摄取迅速，1分钟内硫醇水平稳定。3-巯基丙酸（ζ = -1）的细胞内与细胞外浓度之比（Cin/Cout）为0.74，2-巯基乙醇（ζ = 0）为0.56，半胱胺（ζ = +1）为1.47，WR1065（ζ = +2）为1.04。这些硫醇的kr值分别为：3-巯基丙酸为1.3±0.5×105 dm3mol-1s-1，2-巯基乙醇为3.3±1.6×105 dm3mol-1s-1，半胱胺为3.9±1.1×105 dm3mol-1s-1，WR1065为2.7±1.1×106 dm3mol-1s-1。这些值低于先前发表的关于分离的pBR322 DNA化学修复的值，且随电荷增加的幅度也较小，这可能是因为核蛋白和多胺与大肠杆菌的细胞DNA有关。然而，ζ每增加一个单位，kr大约增加三倍，这表明反离子凝聚和同离子耗尽对于确定带电硫醇在大肠杆菌辐射防护中的有效性很重要。
