INVESTIGADORES
IGLESIAS Alberto Alvaro
artículos
Título:
Purification and Characterization of a Glutathione Reductase from Phaeodactylum tricornutum
Autor/es:
D.G. ARIAS; V.E. MÁRQUEZ; A.J. BECCARIA; S.A. GUERRERO; A.A. IGLESIAS
Revista:
PROTIST
Editorial:
ELSEVIER GMBH
Referencias:
Lugar: Jena; Año: 2010 vol. 161 p. 91 - 101
ISSN:
1434-4610
Resumen:
Glutathionereductase (E.C.1.8.1.7) was purified from Phaeo dactylum tricornutum cells grownaxenically inanautotrophic medium. Theover all 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-and2’,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 toadimer.The enzyme exhibited as pecific activity of 190 Umg1 with an optimal activity at pH8.0 and 32 ºC. We determined Km values of 14 µM and 60 µM 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 an notated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.