Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum.

DIEGO G. ARIAS; VANINA E. MÁRQUEZ; ALEJANDOR J. BECCARIA; SERGIO A. GUERRERO; ALBERTO A. IGLESIAS

Protist

Elsevier

Año: 2010 vol. 161 p. 91 - 101

1434-4610

Glutathione reductase (E.C.1.8.1.7) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2´,5´-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190Umg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14muM and 60muM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.