Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kgs. Lyngby, Denmark.
Phytochemistry. 2010 Oct;71(14-15):1650-6. doi: 10.1016/j.phytochem.2010.06.024. Epub 2010 Aug 18.
Enzymes involved in redox control are important during seed germination and seedling growth. Ascorbate-glutathione cycle enzymes in barley embryo extracts were monitored both by 2D-gel electrophoresis and activity measurements from 4 to 144 h post imbibition (PI). Strikingly different activity profiles were observed. No ascorbate peroxidase (APX) activity was present in mature seeds but activity was detected after 24 h PI and increased 14-fold up to 144 h PI. In contrast, dehydroascorbate reductase (DHAR) activity was present at 4h PI and first decreased by 9-fold until 72 h PI followed by a 5-fold increase at 144 h PI. Glutathione reductase and monodehydroascorbate reductase activities were also detected at 4 h PI, and showed modest increases of 1.8- and 2.7-fold, respectively, by 144 h PI. The combination of functional analysis with the proteomics approach enabled correlation of the activity profiles and protein abundance. While gel spots containing APX showed intensity changes consistent with the activity profile from 0 to 72 h PI, DHAR spot intensities indicated that post-translational regulation may be responsible for the observed changes in activity. Transcript profiling, 2D-western blotting and mass spectrometric characterization of multiple APX spots demonstrated the presence of APX1 and minor amounts of APX2.
参与氧化还原控制的酶在种子萌发和幼苗生长过程中很重要。通过 2D 凝胶电泳和从吸水后 4 到 144 小时（PI）的活性测量监测大麦胚提取物中的抗坏血酸-谷胱甘肽循环酶。观察到明显不同的活性谱。成熟种子中不存在抗坏血酸过氧化物酶（APX）活性，但在 24 h PI 后检测到活性，并在 144 h PI 时增加了 14 倍。相比之下，脱氢抗坏血酸还原酶（DHAR）活性在 4 h PI 时存在，并在 72 h PI 时首先减少了 9 倍，然后在 144 h PI 时增加了 5 倍。谷胱甘肽还原酶和单脱氢抗坏血酸还原酶活性也在 4 h PI 时检测到，在 144 h PI 时分别增加了 1.8 倍和 2.7 倍。功能分析与蛋白质组学方法的结合使得能够将活性谱和蛋白质丰度相关联。虽然含有 APX 的凝胶斑点的强度变化与 0 到 72 h PI 的活性谱一致，但 DHAR 斑点强度表明可能是翻译后调节导致了观察到的活性变化。APX1 和少量 APX2 的存在通过转录谱分析、2D-免疫印迹和多个 APX 斑点的质谱鉴定得到证实。
