Purification and characteristics of glutamate dehydrogenase (GDH) from triticale roots

Abstract

In the investigated 14 day old triticale seedlings a much higher GDH activity was observed in roots than in leaves. The enzyme from the roots was purified up to the state of homogeneity (about 400 fold). The purified enzyme showed a higher activity in the presence of reduced coenzyme forms (NAD(P)H) than their oxidated forms. In the presence of NAD(P)H the enzyme showed absolute specificity to 2-oxoglutarate and in cooperation with NAD(P) + to L-glutamate. The Km values determined for particular substrates indicate a high affinity of NADPH-GDH to ammonium ions.Optimum pH, temperature and thermostability of GDH depended on the type and form of the coenzyme. Molecular mass of purified enzyme was 257 kDa. It seems that native GDH is composed of six identical subunits of the molecular mass 42.5 kDa.

Glutamate dehydrogenase of the germinating triticale seeds: gene expression, activity distribution and kinetic characteristics

“…Gene expression studies confirmed changes in GDH activity dynamics and distribution in triticale seeds. Earlier studies analyzing this enzyme in triticale seedlings also indicated the presence of just one isoform, catalyzing a reversible reaction (Kwinta et al 2001).…”

“…Enzyme activity and protein analysis TsGDH1 were extracted from scutellum and endosperms of seeds imbibed for 8, 16, 24 h and from scutellum, endosperm, shoots and roots of seeds imbibed for 48 and 72 h. Procedure of the enzyme isolation from the triticale seedlings was described earlier (Kwinta et al 2001). GDH activity was determined in both the aminating and the deaminating directions by following the absorption change at 340 nm (Barash et al 1973).…”

“…The GDH protein was isolated and purified from the scutellum of the seeds subjected to 48 h of germination. The procedure published elsewhere (Kwinta et al 2001) has been employed, with one modification: after salt-based precipitation, the samples have been separated by means of HPLC in the Waters Multisolvent Delivery System apparatus. The protein has been partially purified in four steps, including heating the protein suspension at 50°C, ammonium sulfate-derived precipitation (the enzyme was recovered from the fraction between 30 and 60% of the sample saturation) and ionexchange chromatography (anionite) on the 15HR DEAE (1 9 10 cm) column (Waters).…”

Molecular cloning and functional analysis of the second gene encoding glutamate dehydrogenase in triticale

“…The a and b polypeptides combine in different ratios to form seven possible NADH-GDH isoenzymes in A. thaliana, N. plumbaginifolia or V. vinifera (Fontaine et al 2006;Loulakakis and Roubelakis-Angelakis 1991;Masclaux-Daubresse et al 2002). Only one isoform was found to be present in triticale, whereas L. luteus contains fourteen isoforms of this enzyme (Kwinta et al 2001;Ratajczak et al 1986). The quantity and type of GDH isoenzymes in plants depends on the type of tissue, developmental stage and environmental growth conditions.…”

“…The quantity and type of GDH isoenzymes in plants depends on the type of tissue, developmental stage and environmental growth conditions. NADH-dependent enzyme activity is usually higher in roots, developing seeds and ripening fruits compared with that in leaves (Singh and Srivastava 1982;Nanda et al 1991;Kwinta et al 2001). In A. thaliana, the most anodal isoenzyme, i.e., the homohexamer of a subunits, is active in both roots and floral stems, whereas the most cathodal isoenzyme, i.e., the homohexamer of b subunits, is active in leaves (Fontaine et al 2006(Fontaine et al , 2013.…”