Improved procedure for detection of superoxide dismutase isoforms in potato, Solanum tuberosum L.
2014
Тип документа:
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт:
Superoxide dismutase (SOD) in-gel activity assay with selective
inhibitors (KCN and H2O2) is one of the most commonly used methods for
identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and
evaluation of oxidative stress response in plants. However, there are
potential pitfalls that surround this assay, such as problem to detect
isoforms with low activity, comigration of SOD isoforms or application
of inappropriate inhibitor concentration. We propose an improved method
based on the combination of in-gel analysis of SOD activity and
native-PAGE immunoblotting for identification of isoforms and
determination of SOD isoenzyme activity pattern in potato. Depending on
cultivar and growing conditions, one MnSOD, 3 FeSOD and 5-6 Cu/ZnSOD
isoforms were identified in potato leaves. The most important
qualitative difference between ex vitro- and in vitro-grown plants was
the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets
grown in vitro. Compared with results of in-gel activity assay with
selective inhibitors, new method allowed accurate identification of
comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of
ambiguous identities. Potato SODs were also characterized by SDS-PAGE
immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides
(17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were
detected in leaves of four examined cultivars. The difference in the
number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE
and SDS-PAGE immunoblots suggests that SOD proteins may have undergone
post-translational modifications affecting protein mobility or existence
of isoforms that differ from each other in total protein charge, but not
in molecular weight.
Кључне речи:
Potato; Enzyme activity assay; Native-PAGE immunoblottingИзвор:
Acta Physiologiae Plantarum, 2014, 36, 8, 2059-2066
DOI: 10.1007/s11738-014-1583-z
ISSN: 1861-1664