Kaneto H, Fujii J, Suzuki K, Kasai H, Kawamori R, Kamada T, Taniguchi N
Department of Biochemistry, Osaka University Medical School, Japan.
Biochem J. 1994 Nov 15;304 ( Pt 1)(Pt 1):219-25. doi: 10.1042/bj3040219.
Human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) undergoes site-specific and random fragmentation by non-enzymic glycosylation (glycation). Released Cu2+ from the glycated Cu,Zn-SOD probably facilitates a Fenton reaction to convert H2O2 into hydroxy radical, which then participates in the non-specific fragmentation [Ookawara et al. (1992) J. Biol. Chem. 267, 18505-18510]. In the present study, we investigated the effects of glycated Cu,Zn-SOD on cloned DNA fragments and nuclear DNA and analysed the formation of 8-hydroxydeoxyguanosine (8-OH-dG). Incubation of cloned DNA fragments with Cu,Zn-SOD and reducing sugars resulted in cleavage of the DNA. The extent of the cleavage corresponded to the reducing capacity of the sugar. Metal-chelating reagents, EDTA and bathocuproine, and an H2O2 scavenger, catalase, inhibited the DNA cleavage. Hydroxy radical scavengers and aminoguanidine, an inhibitor of glycation, also inhibited the reaction. Moreover, the glycation of Cu,Zn-SOD caused the substantial formation of 8-OH-dG in DNA. When isolated nuclei were incubated with CuCl2 plus H2O2, nuclear DNA cleavage was observed. Incubation of isolated nuclei with Cu,Zn-SOD that had been pre-incubated with glucose also resulted in nuclear DNA cleavage. These results suggest that hydroxy radical is produced through a Fenton reaction by Cu2+ and H2O2 released from the glycated Cu,Zn-SOD, and participates in nuclear DNA cleavage. This mechanism may partly explain the deterioration of organs under diabetic conditions.
人铜锌超氧化物歧化酶（Cu,Zn-SOD）会通过非酶糖基化（糖基化反应）发生位点特异性和随机断裂。糖化的Cu,Zn-SOD释放出的Cu2+可能促进芬顿反应，将H2O2转化为羟基自由基，然后该自由基参与非特异性断裂反应[Ookawara等人（1992年）《生物化学杂志》267卷，18505 - 18510页]。在本研究中，我们研究了糖化的Cu,Zn-SOD对克隆DNA片段和核DNA的影响，并分析了8-羟基脱氧鸟苷（8-OH-dG）的形成。将克隆DNA片段与Cu,Zn-SOD和还原糖一起孵育会导致DNA断裂。断裂程度与糖的还原能力相对应。金属螯合剂乙二胺四乙酸（EDTA）和2,9-二甲基菲咯啉以及过氧化氢清除剂过氧化氢酶可抑制DNA断裂。羟基自由基清除剂和氨基胍（一种糖基化抑制剂）也能抑制该反应。此外，Cu,Zn-SOD的糖基化导致DNA中大量形成8-OH-dG。当分离的细胞核与氯化铜加过氧化氢一起孵育时，可观察到核DNA断裂。将分离的细胞核与预先用葡萄糖孵育过的Cu,Zn-SOD一起孵育也会导致核DNA断裂。这些结果表明，羟基自由基是由糖化的Cu,Zn-SOD释放的Cu2+和H2O2通过芬顿反应产生的，并参与核DNA断裂。这种机制可能部分解释糖尿病条件下器官的恶化。
