Structural and functional analyses of membrane-spanning electron transfer system in neuroendocrine secretory vesicles by utilizing proteoliposome

利用蛋白脂质体分析神经内分泌囊泡跨膜电子传递系统的结构和功能

基本信息

批准号：
12680655
负责人：
TSUBAKI Motonai
金额：
$ 2.18万
依托单位：
Himeji Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. Cytochrome b561Cytochrome b561 exists in the membranes of neuroendocrine vesicles of central and peripheral nervous systems. It functions as a transmembrane electron conduit ; I. e. receiving one electron equivalent from ascorbate at the intravesicular surface and donating one electron equivalent to monodehydro ascorbate radical at the intravesicular surface. We found previously that cytochrome b561 contained two independent heme centers (gz = 3.12 and gz = 3.69 species) by EPR spectroscopy and heme content analysis.2. Redox Potential MeasurementRedox potential measurements of native cytochrome b561 in a detergent-solubilized state showed a presence of two redox components with a different midpoint potential (+155 and +62 mV). DEPC treatment of cytochrome b561 in the oxidized state caused a drastic lowering of the lower redox component as low as -30 mV. This observation is consistent with the notion that the DEPC treatment caused an inhibition of electron accepting ability from asco … More rbate by the specific N-carbethoxylations of conserved residues (His88, His161 and Lys85) of cytochorme b561.3. EPR SpectroscopyEffect of the DEPC-treatment on the oxidized EPR spectrum of cytochrome b561 was examined. However, there was no appreciable difference between the native and the DEPC-treated samples. Upon addition of ascorbate to the native sample, both heme centers could be fully reduced. However, for the DEPC-treated sample, only the gz = 3.12 signal disappeared. The gz = 3.69 signal remained in full intensity. This result suggests that the gz = 3.69 species has a role for the electron accepting from ascorbate at the extravesicular site and is susceptible to the DEPC-modification.4. Transmembrane Electron Transfer Supported by Cytochrome b561Purified cytochrome b561 was successfully reconstituted into ascorbate-loaded vesicle membranes in an inside-out orientation. Cytochrome b561 could donate electron equivalent to extravesicular cytochrome c via transmembrane elecron transfer. Further, cytochrome b561 could support the monooxygenase activity of extravesicular dopamine b-hydroxylase in the absence of any redox mediator. Addition of ferricyanide as a redox mediator enhance the extravesicular monooxygenase acitivity. Less

1.细胞色素b561 细胞色素b561存在于中枢和外周神经系统的神经内分泌囊泡的膜中，其功能是跨膜电子导管；即在囊泡表面接收来自抗坏血酸的一个电子当量并给予单脱氢抗坏血酸自由基。我们之前发现细胞色素 b561 包含两个独立的血红素中心 (gz = 3.12 和 gz = 3.69 种）通过 EPR 光谱和血红素含量分析。 2. 氧化还原电位测量在洗涤剂溶解状态下天然细胞色素 b561 的氧化还原电位测量显示存在两种具有不同中点电位的氧化还原成分（+155 和 +62）。氧化态细胞色素 b561 的 DEPC 处理导致较低的氧化还原成分急剧降低至低至-30 mV。这一观察结果与 DEPC 处理通过细胞色素 EPR 的保守残基（His88、His161 和 Lys85）的特异性 N-碳乙氧基化引起对 asco rbate 电子接受能力的抑制的观点一致。光谱检查了 DEPC 处理对细胞色素 b561 氧化 EPR 光谱的影响，但没有明显差异。在天然样品中添加抗坏血酸后，两个血红素中心都可以完全减少，但是对于 DEPC 处理的样品，仅 gz = 3.12 信号消失，而 gz = 3.69 信号仍然存在。该结果表明，gz = 3.69 物质在囊泡外位点具有接受抗坏血酸电子的作用，并且容易受到 DEPC 修饰。4．细胞色素b561支持的电子转移纯化的细胞色素b561以由内向外的方向成功地重构为负载抗坏血酸的囊泡膜，细胞色素b561可以通过跨膜电子转移提供与囊外细胞色素c相当的电子。此外，细胞色素b561可以支持囊外的单加氧酶活性。多巴胺b-羟化酶在没有任何氧化还原介质的情况下。添加铁氰化物作为氧化还原介体可增强囊泡外单加氧酶的活性。