1. Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei
- Author
-
Kido, Yasutoshi, Sakamoto, Kimitoshi, Nakamura, Kosuke, Harada, Michiyo, Suzuki, Takashi, Yabu, Yoshisada, Saimoto, Hiroyuki, Yamakura, Fumiyuki, Ohmori, Daijiro, Moore, Anthony, Harada, Shigeharu, and Kita, Kiyoshi
- Subjects
- *
TRYPANOSOMA brucei , *AFRICAN trypanosomiasis , *CYTOCHROMES , *TARGETED drug delivery , *ESCHERICHIA coli , *INDUCTIVELY coupled plasma mass spectrometry , *HOST-parasite relationships , *MONOMERS - Abstract
Abstract: 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 338μM and V max of 601μmol/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. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF