Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1
Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1
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DOI：
10.1042/bj20050390
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发表时间：
2005-09-01
影响因子：
4.1
通讯作者：
Aro, EM
中科院分区：
文献类型：
--
作者：
Zhang, PP;Battchikova, N;Aro, EM
蓝藻中的 NDH（NADH-醌氧化还原酶）-1 复合物在呼吸和循环电子流以及活性 CO 吸收中具有特定功能。为了分离NDH-1复合物并研究复合物与复合物之间的相互作用，通过在NDH-1的不同亚基上添加His-标签（组氨酸标签）构建了几种细长热聚球藻菌株。两株在 CupA 和 NdhL 上带有 His 标签的菌株通过一步 Ni2+ 柱色谱成功地分离了 NDH-1 复合物。对 Ni2+ 柱洗脱的蛋白质进行 BN (blue-native)/SDS/PAGE 分析，发现存在三种分子量约为 450、300 和 190 kDa 的复合物，经 MS 鉴定分别为 NDH-1L、NDH-1M 和 NDH-1S，之前在集胞藻中发现。 PCC 6803。还从 NdhL-His 菌株中分离出约 490 kDa 的较大复合物。该复合物被命名为“NDH-1MS”，由 NDH-1M 和 NDH-1S 组成。 NDH-1L 复合物通过 Ni2+ 柱层析从 T. elongatus 的 WT（野生型）细胞中回收。仅存在于 NDH-1L 中的 NdhF1 亚基内部具有 -HHDHHSHH- 序列，该序列似乎对 Ni2+ 柱具有亲和力。通过这种层析没有从WT细胞中回收NDH-1S或NDH-1M。对低浓度去污剂溶解的膜进行 BN/SDS/PAGE 分析表明存在丰富的 NDH-1MS，但不存在 NDH-1M 或 NDH-1S。这些结果清楚地表明NDH-1S在体内与NDH-1M相关。
NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO, uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.