Reconstitution of neuroendocrine vesicles by utilizing proteoliposomes and chromaffin vesicles

利用蛋白脂质体和嗜铬囊泡重建神经内分泌囊泡

基本信息

批准号：
14580676
负责人：
TSUBAKI Motonari
金额：
$ 1.98万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Cytochrome b_<561> from chromaffin vesicles contains two hemes b with different midpoint potentials and participates in transmembrane electron transport from extravesicular ascorbate (AsA) to an intravesicular monooxygenase, dopamine β-hydroxylase. Treatment of oxidized b_<561> with diethylpyrocarbonate (DEPC) caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of AsA during the treatment. EPR analyses showed that, upon the treatment, the g_z=3.69 heme species was converted to a non-AsA-reducible form, although its g_z value showed no appreciable change. The treatment had no effect on the other heme (the g_z=3.13 species). The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the DEPC-treated b_<561> when ASA was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of AsA and increases both the reduction … More rate and the final reduction level of the heme center on the intravesicular side of the DEPC-treated cytochrome. The purified b_<561> was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When AsA-loaded vesicles with b_<561> were mixed with ferricytochrome c, the intravesicular AsA was able to reduce external thiazole blue or cytochrome c. The topology of the reconstituted b_<561> in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that b_<561> was reconstituted into the membranes in an inside-out orientation irrespective of the modification with DEPC. The addition of a soluble form of dopamine β-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon addition of ferricyanide as a mediator between b_<561> and dopamine β-hydroxylase. Less

来自铬脂蔬菜的细胞色素b_ <561>包含两个具有不同中点电势的HEMES B，并参与了从外外抗坏血酸（ASA）到内内注射单氧酶，多巴胺β-羟基酶的跨膜电子转运。用二乙基碳酸盐（DEPC）处理氧化的B_ <561>导致较低分量的中点潜力降档，并且在处理过程中ASA的存在阻止了这种转移。 EPR分析表明，在处理后，G_z = 3.69血红素物种被转化为非ASA可还原形式，尽管其G_z值没有显示可观的变化。治疗对其他血红素没有影响（G_z = 3.13种）。当使用ASA用作还原时，添加亚果仁烷可提高DEPC处理的B_ <561>的最终降低率和最终降低水平。该观察结果表明，亚洲的普遍氧化产生的甲基苯胺清除型自由基自由基，并增加了降低……更多的速率和在DEPC处理的细胞色素静脉内的血红素中心的最终还原水平。纯化的B_ <561>通过dext-diaysis和扩展方法成功地将纯化的B_ <561>成功地重构为胆固醇 - 磷脂酰胆碱 - 磷脂酰甘油蔬菜。当将ASA加载的B_ <561>与铁色素C混合时，静脉内ASA能够减少硫唑外部蓝色或细胞色素c。通过用胰蛋白酶进行处理，然后进行SDS-PAGE和MALDI-TOF-MS分析，检查了囊泡膜中重构B_ <561>的拓扑。在Lys191处只有一个主要的裂解位点，表明B_ <561>在内而外方向以内而外的方向重构，而与DEPC进行了修改无关。在外部培养基中添加固体形式的多巴胺β-羟化酶导致成功重构羟基化活性向酪胺（一种多巴胺的类似物），这表明发生了通过复数形成的直接电子转移。在添加铁氰化物作为B_ <561>和多巴胺β-羟化酶之间的介体时，这种活性显着增强。较少的