Turano F J, Fang T K
United States Department of Agriculture, Agricultural Research Service, Climate Stress Laboratory, Beltsville, Maryland 20705, USA.
Plant Physiol. 1998 Aug;117(4):1411-21. doi: 10.1104/pp.117.4.1411.
Two distinct cDNA clones encoding for the glutamate decarboxylase (GAD) isoenzymes GAD1 and GAD2 from Arabidopsis (L.) Heynh. were characterized. The open reading frames for GAD1 and GAD2 were expressed in Escherichia coli and the recombinant proteins were purified by affinity chromatography. Analysis of the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis suggest that GAD1 and GAD2 encode for 58- and 56-kD peptides, respectively. The enzymatic activities of the pure recombinant GAD1 and GAD2 proteins were stimulated 35- and 13-fold, respectively, by Ca2+/calmodulin but not by Ca2+ or calmodulin alone. Southern-blot analysis of genomic DNA suggests that there is only one copy of each gene in Arabidopsis. The GAD1 transcript and a corresponding 58-kD peptide were detected in roots only. Conversely, the GAD2 transcript and a corresponding 56-kD peptide were detected in all organs tested. The specific activity, GAD2 transcript, and 56-kD peptide increased in leaves of plants treated with 10 mM NH4Cl, 5 mM NH4NO3, 5 mM glutamic acid, or 5 mM glutamine as the sole nitrogen source compared with samples from plants treated with 10 mM KNO3. The results from these experiments suggest that in leaves GAD activity is partially controlled by gene expression or RNA stability. Results from preliminary analyses of different tissues imply that these tendencies were not the same in flower stalks and flowers, suggesting that other factors may control GAD activity in these organs. The results from this investigation demonstrate that GAD activity in leaves is altered by different nitrogen treatments, suggesting that GAD2 may play a unique role in nitrogen metabolism.
对来自拟南芥（Arabidopsis (L.) Heynh.）的编码谷氨酸脱羧酶（GAD）同工酶GAD1和GAD2的两个不同cDNA克隆进行了表征。GAD1和GAD2的开放阅读框在大肠杆菌中表达，重组蛋白通过亲和层析纯化。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和免疫印迹分析对重组蛋白进行分析，结果表明GAD1和GAD2分别编码58 kD和56 kD的肽。纯重组GAD1和GAD2蛋白的酶活性分别被Ca2+/钙调蛋白刺激35倍和13倍，但单独的Ca2+或钙调蛋白则无此作用。基因组DNA的Southern印迹分析表明，拟南芥中每个基因只有一个拷贝。仅在根中检测到GAD1转录本和相应的58 kD肽。相反，在所有测试器官中均检测到GAD2转录本和相应的56 kD肽。与用10 mM KNO3处理的植物样品相比，以10 mM NH4Cl、5 mM NH4NO3、5 mM谷氨酸或5 mM谷氨酰胺作为唯一氮源处理的植物叶片中，GAD2的比活性、转录本和56 kD肽均增加。这些实验结果表明，在叶片中GAD活性部分受基因表达或RNA稳定性的控制。对不同组织的初步分析结果表明，这些趋势在花茎和花中并不相同，这表明其他因素可能控制这些器官中的GAD活性。本研究结果表明，不同的氮处理会改变叶片中的GAD活性，这表明GAD2可能在氮代谢中发挥独特作用。
