Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 57, Issue 21, Pages 10357-10362Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jf9021256
Keywords
Taiwanofungus camphorata; glutaredoxin; three-dimension homology structure (3D homology structure); expression; beta-hydroxyethyl disulfide [HED, (HOCH2CH2)(2)S-2]; dehydroascorbate (DHA)
Funding
- National Science Council of the Republic of China [NSC 97-2313-B-019-001-MY3]
Ask authors/readers for more resources
Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli. Functional TcGrx was expressed and purified by Ni2+-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of similar to 15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (K-m) values for beta-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 degrees C was 8.5 min, and its thermal inactivation rate constant K-d was 6.52 x 10(-2) min(-1). The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available