Jasid Sebastián, Simontacchi Marcela, Bartoli Carlos G, Puntarulo Susana
Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina.
Plant Physiol. 2006 Nov;142(3):1246-55. doi: 10.1104/pp.106.086918. Epub 2006 Sep 15.
Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.
通过电子顺磁共振自旋捕捉技术，对大豆（Glycine max var. ADM 4800）叶绿体产生一氧化氮（NO）作为内源性产物进行了研究。亚硝酸盐和L-精氨酸（Arg）是被认为可能是植物中NO来源的酶促活性的底物。在存在1 mM亚硝酸钠（NaNO₂）的情况下，大豆叶绿体显示出NO产生速率为3.2±0.2 nmol min⁻¹ mg⁻¹蛋白质。3-(3,4-二氯苯基)-1,1-二甲基脲对光合电子流的抑制导致NO产生速率降低（1.21±0.04 nmol min⁻¹ mg⁻¹蛋白质）。用1 mM Arg孵育的叶绿体显示出NO产生速率为0.76±0.04 nmol min⁻¹ mg⁻¹蛋白质，这一速率不受孵育培养基中Ca²⁺缺失或添加Ca²⁺和钙调蛋白的影响。当叶绿体在一氧化氮合酶抑制剂N(ω)-硝基-L-精氨酸甲酯盐酸盐和N(ω)-硝基-L-精氨酸存在下孵育时，这种产生受到抑制。叶绿体在体外暴露于NO供体（250 μM S-亚硝基谷胱甘肽）会使膜中的脂质自由基含量降低29%；然而，在存在25 μM过氧亚硝酸根（ONOO⁻）的情况下孵育会导致脂质衍生自由基增加（34%）。通过蛋白质免疫印迹法测定了ONOO⁻对蛋白质氧化的影响，结果表明与对照相比，基质或类囊体蛋白中的羰基含量增加。此外，ONOO⁻处理显著影响类囊体中的氧气释放和叶绿素荧光。此处报道的数据表明，NO是大豆叶绿体中的内源性代谢物，活性氮物种可能对叶绿体大分子发挥抗氧化或促氧化作用。
