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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680655/
• 关键词: Vitamin C; ascorbic acid; cytochrome b561; noradrenaline; catecholamine; chromaffin vesicle; transmembrane electron transfer; heme; RecJ蛋白質; 高度好熱菌

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环形B561存在于中央和周围神经系统的神经内分泌蔬菜的膜中。它充当跨膜电子导管； IE。从静脉内表面上接收一个电子等效的电子等效物，并在静脉内表面捐赠一个电子等效于单螺旋藻抗坏血酸。我们以前发现，通过EPR光谱和血红素含量分析，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）2。在确定的溶解状态下，天然细胞色素B561的氧化还原电势测量值测量表明存在两个具有不同中点电位的氧化还原分量（+155和+62 mV）。 DEPC在氧化状态下对细胞色素B561的处理导致下层氧化还原成分急剧低至-30 mV。该观察结果与DEPC处理导致ASCO的电子接受能力的抑制作用相一致。 DEPC处理对细胞色素B561的氧化EPR光谱的EPR光谱效应。但是，天然和DEPC处理的样品之间没有明显的差异。在向天然样品中添加抗坏血酸酯后，两个血红素中心都可以完全降低。但是，对于经DEPC处理的样品，仅GZ = 3.12信号消失了。 GZ = 3.69信号保持完全强度。该结果表明，GZ = 3.69种对电子在奢华部位接受的电子接受，并且容易受到DEPC模型的影响。4。由细胞色素B561PURIFIED的细胞色素B561支持的跨膜电子转移成功地重构为抗坏血酸的囊泡膜，以内而外的方向重组。细胞色素B561不能通过跨膜电染色体转移等效于奢侈的细胞色素c。此外，在没有任何氧化还原介质的情况下，细胞色素B561可以支持局外多巴胺B-羟化酶的单加氧酶活性。添加铁酰亚胺作为氧化还原介质增强了外外单（单加掺杂酶的活性）。较少的

项目成果

Neya S., Tsubaki M., Hori H., Yonetani T., and Funasaki N.: "Unusual spin state equilibrium of azide metmyoglobi induced by core deformed heme"Inorganic Chemistry. 40. 1220-1225 (2001)

Neya S.、Tsubaki M.、Hori H.、Yonetani T. 和 Funasaki N.：“核心变形血红素诱导的叠氮化物metmyoglobi 的异常自旋态平衡”无机化学。

Asada A., Orii H., Agata K., Watanabe K., and Tsubaki M.: "Planarian cvto6hrome b561 : Conservation of a six transmembrane structure and localization along the central and peripheral nervous system"Journal o Biochemistry. 131. 175-182 (2002)

Asada A.、Orii H.、Agata K.、Watanabe K. 和 Tsubaki M.：“涡虫 cvto6hrome b561：六跨膜结构的保守以及中枢和周围神经系统的定位”《生物化学杂志》。

Atsushi Yamagata: "The Crystal Structure of Exonuclease RecJ Bound to Mn^<2+>Ion Suggests How Its Characteristic Motifs Are Involved in Exonuclease Activity"Proc. Natl. Acad. Sci. USA. (in press).

Atsushi Yamagata：“与 Mn^<2> 离子结合的核酸外切酶 RecJ 的晶体结构表明其特征基序如何参与核酸外切酶活性”Proc。

Tsubaki M., Kobayashi K., Ichise T., Takeuchi F., and Tagawa S.: "Diethylpyrocarbonate-modification abolishes fast electron accepting ability of cytochrome b561 from ascorbate but does not influence on electron donation to monodehydroascorbate radical : I

Tsubaki M.、Kobayashi K.、Ichise T.、Takeuchi F. 和 Takawa S.：“焦碳酸二乙酯修饰消除了抗坏血酸细胞色素 b561 的快速电子接受能力，但不影响单脱氢抗坏血酸自由基的电子供给：I

Takeuchi T., Tsubaki M., Futagawa J., Masuya F., and Hori H.: "Adrenodoxin-cytochrome P450scc interaction as revealed by EPR spectroscopy : Comparison with putidaredoxin-cytochrome P450cam system"Journal of Biochemistry. 30. 789-797 (2001)

Takeuchi T.、Tsubaki M.、Futakawa J.、Masuya F. 和 Hori H.：“EPR 光谱法揭示的肾上腺氧还蛋白-细胞色素 P450scc 相互作用：与 putidaredoxin-细胞色素 P450cam 系统的比较”生物化学杂志。