Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.
Multiple complexes of nitrogen assimilatory enzymes in spinach chloroplasts: possible mechanisms for the regulation of enzyme function.
复制标题
DOI：
10.1371/journal.pone.0108965
复制
发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Hase T
中科院分区：
文献类型：
--
作者：
Kimata-Ariga Y;Hase T
氮的同化是植物生长和生产力的重要生物过程。通过蓝色原生电泳（BN-PAGE）的western blots分析，我们发现菠菜叶绿体中参与氮同化的三种叶绿体酶——谷氨酸合成酶（GOGAT）、亚硝酸盐还原酶（NiR）和谷氨酰胺合成酶（GS）分别组装成不同的蛋白质复合物。GOGAT和NiR不仅以单体形式存在，而且在叶绿体基质组分中分别以离散大小（730 kDa）和多重大小（bb0 - 120 kDa）的新型复合物形式存在。这些配合物在BN-PAGE后的二维SDS-PAGE上显示出与每个单体相同的迁移率。含有GOGAT的730kda配合物随着基质溶剂pH的变化解离成单体，多个近红外配合物可逆地转化为单体。另一方面，抗gs抗体检测到的条带不仅作为420 kDa的传统十聚体全酶复合物存在于基质中，而且作为560 kDa的新型复合物存在于类囊体中。在2D SDS-PAGE上，560 kDa复合物中的多肽的迁移速度比420 kDa复合物慢，这意味着不同GS亚型的组装或相同GS蛋白的翻译后修饰。这些复合物的功能是通过天然条件下凝胶内GS活性以及NiR和GOGAT与其生理电子供体铁氧还蛋白的结合能力来评估的。结果表明，三种氮同化酶在大小和位置上的多样性可能与酶功能的生理调节有关，与最近描述的碳同化酶的情况类似。
Assimilation of nitrogen is an essential biological process for plant growth and productivity. Here we show that three chloroplast enzymes involved in nitrogen assimilation, glutamate synthase (GOGAT), nitrite reductase (NiR) and glutamine synthetase (GS), separately assemble into distinct protein complexes in spinach chloroplasts, as analyzed by western blots under blue native electrophoresis (BN-PAGE). GOGAT and NiR were present not only as monomers, but also as novel complexes with a discrete size (730 kDa) and multiple sizes (>120 kDa), respectively, in the stromal fraction of chloroplasts. These complexes showed the same mobility as each monomer on two-dimensional (2D) SDS-PAGE after BN-PAGE. The 730 kDa complex containing GOGAT dissociated into monomers, and multiple complexes of NiR reversibly converted into monomers, in response to the changes in the pH of the stromal solvent. On the other hand, the bands detected by anti-GS antibody were present not only in stroma as a conventional decameric holoenzyme complex of 420 kDa, but also in thylakoids as a novel complex of 560 kDa. The polypeptide in the 560 kDa complex showed slower mobility than that of the 420 kDa complex on the 2D SDS-PAGE, implying the assembly of distinct GS isoforms or a post-translational modification of the same GS protein. The function of these multiple complexes was evaluated by in-gel GS activity under native conditions and by the binding ability of NiR and GOGAT with their physiological electron donor, ferredoxin. The results indicate that these multiplicities in size and localization of the three nitrogen assimilatory enzymes may be involved in the physiological regulation of their enzyme function, in a similar way as recently described cases of carbon assimilatory enzymes.
登录
查看更多内容
DOI：
10.1107/s1399004713034718
发表时间：
2014-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Torreira E;Seabra AR;Marriott H;Zhou M;Llorca Ó;Robinson CV;Carvalho HG;Fernández-Tornero C;Pereira PJ
通讯作者：
Pereira PJ
影响因子：
7.2
作者：
Juric, Snjezana;Hazler-Pilepic, Kroata;Fulgosi, Hrvoje
通讯作者：
Fulgosi, Hrvoje
影响因子：
4.8
作者：
Masalkar, Pintu;Wallace, Ian S.;Roberts, Daniel M.
通讯作者：
Roberts, Daniel M.
影响因子：
4.8
作者：
Petoukhov, MV;Svergun, DI;Vanoni, MA
通讯作者：
Vanoni, MA
影响因子：
2.9
作者：
Swamy, U;Wang, MT;Allen, JP
通讯作者：
Allen, JP