title: Participation of thioredoxin in the V(V)-reduction reaction by Vanabin2.
abstract: BACKGROUND
  It is well-understood that ascidians accumulate high levels of vanadium, a reduced form of V(III), in an extremely acidic vacuole in their blood cells. Vanabins are small cysteine-rich proteins that have been identified only from vanadium-rich ascidians. A previous study revealed that Vanabin2 can act as a V(V)-reductase in the glutathione cascade.
  
  
  METHODS
  AsTrx1, a thioredoxin gene, was cloned from the vanadium-rich ascidian, Ascidia sydneiensis samea, by PCR. AsTrx1 and Vanabin2 were prepared as recombinant proteins, and V(V)-reduction by Vanabin2 was assessed by ESR and ion-exchange column chromatography. Site-directed mutagenesis was performed to examine the direct involvement of cysteine residues. Tissue expression of AsTrx1 was also examined by RT-PCR.
  
  
  RESULTS
  When reduced AsTrx1 and Vanabin2 were combined, Vanabin2 adopted an SS/SH intermediate structure while V(V) was reduced to V(IV). The loss of cysteine residues in either Vanabin2 or AsTrx1 caused a significant loss of reductase activity. Vapp and Kapp values for Vanabin2-catalyzed V(V)-reduction in the thioredoxin cascade were 0.066mol-V(IV)/min/mol-Vanabin2 and 0.19mM, respectively. The Kapp value was 2.7-fold lower than that observed in the glutathione cascade. The AsTrx1 gene was expressed at a very high level in blood cells, in which Vanabins 1-4 were co-expressed.
  
  
  CONCLUSIONS
  AsTrx1 may contribute to a significant part of the redox cascade for V(V)-reduction by Vanabin2 in the cytoplasm of vanadocytes, but prevails only at low V(V) concentrations.
  
  
  CONCLUSIONS
  This study is the first to report the reduction of V(V) in the thioredoxin cascade.
authors.affiliation: Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan; Marine Biological Laboratory, Graduate School of Science, Hiroshima University, 2445 Mukaishima, Onomichi, Hiroshima 722-0073, Japan. Electronic address: ueki@hiroshima-u.ac.jp.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ueki
authors.affiliation: Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Uwagaki
authors.affiliation: Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Sohei
authors.initials: S
authors.lastname: Yamamoto
authors.affiliation: Molecular Physiology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan; Hiroshima University, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Michibata
keywords: Ascidian; Metal; Redox; Reductase; Vanadium