Purification and characterization of a glutathione reductase from Phaeodactylum tricornutum.
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 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14 microM and 60 microM 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.
Arias
Diego G
DG
Laboratorio de Bioquímica Microbiana, Instituto de Agrobiotecnología del Litoral (IAL; FBCB, UNL-CONICET), Paraje El Pozo CC 242, Santa Fe S3000ZAA, Argentina.
Marquez
Vanina E
VE
Beccaria
Alejandro J
AJ
Guerrero
Sergio A
SA
Iglesias
Alberto A
AA
D020418:Algal Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000645:Ammonium Sulfate / Q000378:metabolism; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017377:Diatoms / Q000201:enzymology*; D019281:Dimerization; D004795:Enzyme Stability; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D013696:Temperature
D020418:Algal Proteins; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione; D000645:Ammonium Sulfate