Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica
Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica
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DOI：
10.1104/pp.105.064352
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发表时间：
2005-08-01
期刊：
影响因子：
7.4
通讯作者：
Yazaki, K
中科院分区：
文献类型：
--
作者：
Otani, M;Shitan, N;Yazaki, K
生物碱是植物次生代谢产物中最大的一类。它们中的许多具有很强的生物活性，并且在大多数情况下，它们在合成后积累在产生物碱植物的中央液泡中。然而，生物碱在液泡膜中转运的机制还知之甚少。本研究分析了异喹啉类生物碱小檗碱在黄连培养细胞液泡中产生和积累的液泡转运机制。小檗碱运输使用完整的液泡和液泡膜囊泡系统的表征表明，小檗碱的吸收刺激Mg/ATP，以及GTP，CTP，UTP，和Mg/焦磷酸。小檗碱摄取强烈抑制NH 4+和巴弗洛霉素A1，而钒酸盐，这是常用的抑制ATP结合盒转运，只有轻微的影响，这表明存在一个典型的二级运输机制。这与这种植物细胞质膜中的情况相反，其中ATP结合盒转运蛋白参与小檗碱转运。脂质体模型实验表明，离子阱机制几乎没有牵连在小檗碱的运输。进一步的研究表明，小檗碱通过H+/小檗碱反向转运蛋白，它具有43.7 μ M的小檗碱的Km值跨液泡膜运输。竞争实验使用各种小檗碱类似物，以及其他类别的生物碱，揭示了这种转运是相当具体的，但不是排他性的，小檗碱。
Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4+ and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a K-m value of 43.7 mu M for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.