On the cross-roads of main carbon and nitrogen metabolic pathways, glutamate dehydrogenase (GDH, E.C. 1.4.1.2) carries out the reaction of reductive amination of 2-oxoglutarate to glutamate (the anabolic activity; NAD(P)H-GDH), and the reverse reaction of oxidative deamination of glutamic acid (the catabolic activity; NAD(P)? -GDH). To date, there have been no reports on identification of GDH genes in cereals. Here, we report cloning and biochemical characterization of the GDH from germinating triticale seeds, a common Polish cereal. A single TsGDH1 gene is 1,620 bp long, while its 1,236 bp long open reading frame encodes a protein of 411 amino acids of high homology with the published GDH protein sequences from other plants. Phylogenetic analyses locate the TsGDH1 among other monocotyledonous proteins and among the sequences of the b-type subunit of plant GDHs. Changes in TsGDH1 expression and the dynamics of enzyme activity in germinating seeds confirm the existence of one TsGDH isoform with varying expression and activity patterns, depending on the tissue localization and stage of germination. The four-step purification method (including the anionite chromatography using HPLC) resulted in a protein preparation with a high-specific activity and purification factor of approx. 230. The purified enzyme exhibited an absolute specificity towards 2-oxoglutarate (NAD(P)H-GDH), or towards L-glutamate in the reverse reaction (NAD(P)? -GDH), while its low K m constants towards all substrates and co-enzymes may suggest its aminating activity during germination, or, alternatively, its capability to adjust the direction of the catalyzed reaction according to the metabolic necessity.

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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.

In higher plants, glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.2) are the predominant enzymes in nitrogen metabolism. In this study, we cloned both the GS and GDH genes and analyzed their expression levels and variations in their activity in developing and germinating x Triticosecale (cv. Witon) kernels. The developing kernel samples were collected 3, 5, 7, 9, 13, 15, 20, 25, 30, 35, 40 and 45 days after flowering (DAF). The germinating kernel samples were collected after 8, 16, 24, 48 and 72 h of imbibition. There are two GS isoforms that are localized to different compartments: the cytosol (GS1) and the chloroplast (GS2). Five cDNAs encoding GS proteins in triticale plants were obtained using RT-PCR. We cloned the four genes encoding GS1, which we designated TsGS1-1, TsGS1-2, TsGS1-3 and TsGS1-4 and the only gene encoding GS2, which was designated TsGS2-1. We studied the changes in the enzymatic activity and the expression profiles of the GDH, GS1 and GS2 genes in both the developing and germinating seeds of triticale. Based on our results, there is likely cooperation between GDH and GS1 in the synthesis of glutamine and glutamate during the early stages of seed formation and in the scutella of kernels for up to 24 h of imbibition.

De part ment of Bio chem is try, Ag ri cul tural Uni ver sity of War saw, Key words: cad mium, am mo nia as sim i la tion, glutamine synthetase, glu ta mate dehydrogenase, isoenzymes, triticale Ab stractThe stud ies were per formed on young triticale seed lings grown on a min eral me dium con tain ing 5 mM NO 3 -as the ni tro gen source, with the ad di tion of 0.5 mM CdCl 2. It was de ter mined that cad mium ions ac cu mu lated mainly in the plant roots. Decreases in ni trate con cen tra tions both in the roots and shoots of seed lings, as well as de creases in sol u ble pro tein con tents with si mul ta neous in creases in endopeptidase ac tiv ity were also observed. Both in roots and shoots sig nif i cant de creases in glutamic acid were noted. Toxic cad mium ion ac cu mu la tion in seed lings sig nif i cantly mod i fied ac tiv ity of pri mary ni tro gen as sim i lat ing en zymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glu ta mate dehydrogenase (GDH, EC 1.4.1.2). There was a sig nif i cant de crease in GS ac tiv ity both in roots and in shoots of the stressed plants, in com par i son to plants grown with out cad mium. In shoots of the con trol plants and plants sub jected to stress two GS isoforms were dis cov ered: cytoplasmatic (GS 1 ) and chloroplastic (GS 2 ). Sub stan tial decreases in to tal glutamine synthetase ac tiv ity in green parts of seed lings, oc cur ring un der stress con di tions, re sult from dramatic de crease in GS 2 ac tiv ity (by 60 % in re la tion to the control plants); de spite si mul ta neous in creases in the cytoplasmatic isoform (GS 1 ) ac tiv ity by approx. 96 %.Cad mium ions ac cu mu lat ing in roots and shoots of seed lings not only in creased GDH ac tiv ity, but also mod i fied its coenzymatic spec i fic ity.