2010年4月
Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
- 巻
- 1797
- 号
- 4
- 開始ページ
- 443
- 終了ページ
- 450
- 記述言語
- 英語
- 掲載種別
- DOI
- 10.1016/j.bbabio.2009.12.021
- 出版者・発行元
- ELSEVIER SCIENCE BV
The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338 mu M and V(max) of 601 mu mol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1. (C) 2009 Elsevier B.V. All rights reserved.
- リンク情報
- ID情報
-
- DOI : 10.1016/j.bbabio.2009.12.021
- ISSN : 0005-2728
- eISSN : 1879-2650
- CiNii Articles ID : 80020845577
- Web of Science ID : WOS:000275657200002