Abstract
Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for other plant species. GDH protein is a hexamer composed, in the case of Arabidopsis, of a combination of GDHα, GDHβ and GDHγ subunits. Every combination of subunits is possible; however, it is still barely known whether different combinations affect the enzymatic properties of the hexamers. In other species, hexamers are a combination of GDHα and GDHβ but it cannot be discarded the existence of other genes since for instance GDHγ subunit in Arabidopsis was described in Fontaine et al. (2012).Glutamate + NAD+ + H+ → 2-Oxoglutarate + NADH + NH4+
Keywords: Glutamate dehydrogenase, Enzyme activity, Spectrophotometry
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Recipes
Note: All solutions should be made in ultrapure water.
Acknowledgments
This protocol is adapted from Sarasketa et al. (2014) and based on the methodology reported by Groat and Vance (1981). This work was supported by the Basque Government (IT526-10), the UPV/EHU (EHUA14/14), the People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program (FP7/2007–2013) under REA grant agreement number 334019 and MINECO (BIO2014-56271-R).
References
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As described in Procedure, point D. It is usual to determine the activity out of 20 microliters of extract adding 280 microliters of reaction buffer. However, in function of the species, the tissue and the developmental stage GDH activity is different. Thus, if the activity is very high you may need to dilute the sample to obtain a linear kinetic. On the contrary, if it is too low you may need to concentrate the sample.