Аннотация
Background: Peroxiredoxins have diverse functions in cellular
defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2
and alkyl-hydroperoxide. This study describes the purification and
characterization of a genuine 2-Cys-Prx from Vigna unguiculata
(Vu-2-Cys-Prx).
Methods: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate
fractionation, chitin affinity and ion exchange chromatography.
Results: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is
a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that
appears as a 22 kDa molecule under reducing conditions, indicating that
it is a homodimer linked intermolecularly by disulfide bonds and has a
pI range of 4.56-4.72; its NH2-terminal sequence was similar to
2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%).
Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622
kDa/5.18. Vu-2-Cys-Prx has 8% alpha-helix, 39% beta-sheet, 22% of
turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has
optimal activity at pH 7.0, and prevented plasmid DNA degradation.
Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers
which might be associated with its molecular chaperone activity that
prevented denaturation of insulin and citrate synthase. Its cDNA
analysis showed that the redox-active Cys(52) residue and the amino
acids Pro(45), Thr(49) and Arg(128) are conserved as in other 2-Cys-Prx.
General significance: The biochemical and molecular features of
Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date,
only one publication reported on the purification of native 2-Cys-Prx
from leaves and the subsequent analysis by N-terminal Edman sequencing,
which is crucial for construction of stromal recombinant 2-Cys-Prx
proteins. (C) 2012 Elsevier B.V. All rights reserved.
Пользователи данного ресурса
Пожалуйста,
войдите в систему, чтобы принять участие в дискуссии (добавить собственные рецензию, или комментарий)