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Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis   

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Original research article

A brief version of this protocol appeared in:
Physiologia Plantarum
Feb 2009

Abstract

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyzes the third and irreversible reaction of the pentose phosphate pathway (PPP). It carries out the oxidative decarboxylation of the 6-phosphogluconate to yield ribulose-5-phosphate, carbon dioxide and NADPH. In higher plants, 6PGDH has several subcellular localizations including cytosol, chloroplast, mitochondria and peroxisomes (Corpas et al., 1998; Krepinsky et al., 2001; Mateos et al., 2009; Fernández-Fernández and Corpas, 2016; Hölscher et al., 2016). Using Arabidopsis thaliana as plant model and sweet pepper (Capsicum annuum L.) fruits as a plant with agronomical interest, this protocol illustrates how to prepare the plant extracts for the separation of the potential 6PGDH isoforms by electrophoresis on 6% polyacrylamide non-denaturing gels. Thus, this method allows detecting three 6PGDH isoforms in Arabidopsis seedlings and two 6PGDH isoforms in sweet pepper fruits.

Keywords: NADPH, Non-denaturing polyacrylamide gel electrophoresis, Pentose phosphate pathway, 6-Phosphogluconate dehydrogenase

Background

Non-denaturing gel electrophoresis is a powerful technique that allows separating native proteins. Their mobility depends of protein size, shape and net charge. In these analytical conditions, the protein preserves its activity and in combination with a specific staining method, it allows to separate the presence of potential isoforms. This approach has been widely used in the case of the family of superoxide dismutases. However, to our knowledge, there are not many papers that analyze the presence of different isoforms of 6PGDH activity in plant tissues (Corpas et al., 1998; Mateos et al., 2009). This straight method may be very useful for researchers working with plant 6PGDHs.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Corpas, F. J., de Freitas-Silva, L., García-Carbonero, N., Contreras, A., Terán, F., Ruíz-Torres, C. and Palma, J. M. (2017). Separation of Plant 6-Phosphogluconate Dehydrogenase (6PGDH) Isoforms by Non-denaturing Gel Electrophoresis. Bio-protocol 7(14): e2399. DOI: 10.21769/BioProtoc.2399.
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