Reversely-Oriented Cytochrome b561, in Reconstituted Vesicles Catalyzes Transmembrane Electron Transfer and Supports Extravesicular Dopamine β-Hydroxylase Activity

• Seike Yumiko

  Department of Life Science, Graduate School of Science, Himeji Institute of Technology

• Takeuchi Fusako

  Department of Life Science, Graduate School of Science, Himeji Institute of Technology

• Tsubaki Motonari

  Department of Life Science, Graduate School of Science, Himeji Institute of Technology GREST, JST Department of Molecular Science, Graduate School of Science and Technology, Kobe University

書誌事項

タイトル別名

• Reversely-Oriented Cytochrome <i>b</i>₅₆₁ in Reconstituted Vesicles Catalyzes Transmembrane Electron Transfer and Supports Extravesicular Dopamine β-Hydroxylase Activity
• Reversely Oriented Cytochrome b561 in Reconstituted Vesicles Catalyzes Transmembrane Electron Transfer and Supports Extravesicular Dopamine ベータ Hydroxylase Activity
• Reversely-Oriented Cytochrome b561 in Reconstituted Vesicles Catalyzes Transmembrane Electron Transfer and Supports Extravesicular Dopamine  -Hydroxylase Activity
• Reversely-oriented cytochrome b561 in reconstituted vesicles catalyzes transmembrane electron transfer and supports the extravesicular dopamine β-hydroxylase activity

抄録

Cytochrome b₅₆₁ from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b₅₆₁ can donate electron equivalents directly to cytochrome c. The purified cytochrome b₅₆₁ was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b₅₆₁ were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b₅₆₁ in the vesicle membranes. Pre-treatment of cytochrome b₅₆₁ with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b₅₆₁ in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleav-age site at Lys191 was identified, indicating that cytochrome b₅₆₁ was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. 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 the addition of ferricyanide as a mediator between cytochrome b₅₆₁ and dopamine β-hydroxylase.

収録刊行物

• The Journal of Biochemistry

  The Journal of Biochemistry 134 (6), 859-867, 2003

  社団法人 日本生化学会

キーワード

• ascorbate
• cytochrome <i>b</i>₅₆₁
• dopamine β-hydroxylase
• reconstituted vesicle
• transmembrane electron transfer

詳細情報

• CRID: 1390001204965225344
• NII論文ID: 130003534638
• NII論文ID: 10012059922
• NII書誌ID: AA00694073
• DOI: 10.1093/jb/mvg212
• COI: 1:CAS:528:DC%2BD2cXis1Kks70%3D
• ISSN: 17562651
• ISSN: 0021924X
• NDL書誌ID: 6830823
• PubMed: 14769875
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I6830823
• Web Site: http://academic.oup.com/jb/article-pdf/134/6/859/2380965/mvg212.pdf
• Web Site: https://search.jamas.or.jp/link/ui/2004130090
• 本文言語コード: en
• データソース種別:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
• 抄録ライセンスフラグ: 使用不可