Immunocharacterization of NADH-Glutamate Dehydrogenase from Vitis vinifera L

Abstract

Rabbit antiserum was raised against NADH-glutamate dehydrogenase (GDH) isoenzyme 1, purified from leaves of Vitis vinifera L. cv Soultanina and its specificity was tested. This antiserum was used for immunocharacterization of the GDH from leaf, shoot, and root tissues. The antiserum recognized the seven isoenzymes of NADH-GDH and precipitated all the enzyme activity from the three tissues tested. Westem blot following SDS-PAGE revealed the same protein band for the three tissues, with a molecular mass of 42.5 kilodaltons corresponding to NADH-GDH subunit. Results, based on the immunological studies, revealed that NADH-GDH from leaf, shoot, and root tissues are closely related proteins. Furthermore, addition of ammonium ions to the culture medium of in vitro grown explants resulted in a significant increase in NADH-GDH activity in root, shoot, and leaf tissues.The physiological role of large amounts of GDH' (EC 1.4.1.2.) present in the tissues of higher plants is still obscure. Possible conditions under which GDH may play a significant role in cell metabolism have not been fully resolved.One function of mitochondrial GDH is to synthesize glutamate from some of the ammonia ions released within the mitochondrion by glycine decarboxylation during photorespiration (17,18).The enzyme present in different tissues may be different in its isoenzymic pattern, in its regulatory properties, and control mechanism. In Medicago, GDH organ-specific isoenzymes were reported (8) and in Zea root, callus, and leaves, different isoenzymes/conformers of GDH were found (6). By using immunochemical approach, antigenic homologies between GDH forms from pea seeds were detected (10). In addition, exogenous nitrogen source has been reported to affect the in vivo and in vitro GDH activity (6, 7, 15 and references therein); however, there is not yet enough information on the molecular mechanisms of GDH regulation in higher plants.Recently, NADH-GDH from grapevine (Vitis vinifera L. cv Soultanina) was characterized and its major isoenzyme was purified 2050-fold to homogeneity. The molecular mass of the native enzyme was estimated to be 252 kD and it consisted of six identical 42.5 kD subunits. The amination reaction was fully activated by about 100 Mm Ca2" while the deamination reaction was not affected by the addition of Ca2+ (4).'Abbreviation: GDH, glutamate dehydrogenase.In this report we present further results on the preparation and characterization of GDH-specific antibodies raised against the NADH-GDH isoenzyme 1 from grapevine leaf tissue and on the immunological characteristics of leaf, shoot, and root GDH from the same plant species. In addition, we present information on the effect of nitrogen source on NADH-GDH in in vitro grown grapevine plantlets. MATERIALS AND METHODS Plant MaterialVitis vinifera L. cv Soultanina in vitro grown plants were used. One node explants from green shoots of in vitro grown vines were aseptically positioned for rooting into Roubelakis medium (13), and were kept in a tissue culture room at 25 + 1...

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

“…Analytical and preparative SDS-PAGE of protein extracts was performed as described by Loulakakis and RoubelakisAngelakis (1992). Transfer of electrophoretically resolved proteins onto nitrocellulose filters (0.2-mm pore size; Schleicher and Schuell) was performed according to Loulakakis and Roubelakis-Angelakis (1990b). GDH isoenzymes were localized in nondenaturating polyacrylamide gels using the in situ staining technique as analytically described by Loulakakis and Roubelakis-Angelakis (1991).…”

Glutamate Dehydrogenase of Tobacco Is Mainly Induced in the Cytosol of Phloem Companion Cells When Ammonia Is Provided Either Externally or Released during Photorespiration

“…For immunotransmission electron microscopy studies, ultra thin sections were mounted on 400-mm mesh nickel grids and allowed to dry at 37°C. Sections were first incubated with 5% (v/v) normal goat serum in T1 buffer (0.05 M Tris-HCl buffer containing 2.5% [w/v] NaCl, 0.1% [w/v] BSA, and 0.05% [v/v] Tween 20, pH 7.4) for 1 h at room temperature then with anti-GDH rabbit serum (Loulakakis and Roubelakis-Angelakis, 1990) diluted 70 times in T1 buffer for 6 h at room temperature. Sections were then washed 5 times with T1 buffer, 2 times with T2 buffer (0.02 M Tris-HCl buffer containing 2% [w/v] NaCl, 0.1% [w/v] BSA, and 0.05% [v/v] Tween 20, pH 8) and incubated for 2 h at room temperature with 10 nm colloidal gold-goat anti-rabbit immunoglobulin complex (Sigma, St. Louis) diluted 50 times in T2 buffer.…”

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

“…The mitochondrial marker enzyme fumarase and the peroxisomal / glyoxysomal marker enzyme Cat were assayed as described by Hill and Bradshaw (1969) and Beers and Sizer (1952) (Newman et al, 1994), and the expressed sequence tag clones 137024T7 (T46178) and 188123T7 (H37750) were obtained from the Arabidopsis Biological Resource Center. Rabbit serum raised against grape leaf NAD(H)-GDH (Loulakakis and Roubelakis-Angelakis, 1990b) was used to screen the cDNA expression library as described by Young and Davis (1983). Putative GDH clones were sequenced by the dideoxy chain-termination method using modified T7 DNA polymerase (Sequenase 2.0, United States Biochemical) as described by the manufacturer.…”

“…Immunoblot analysis was conducted as described by Turano et al (1990); proteins were resolved in a SDSpolyacrylamide (8.0%) gel (Laemmli, 1970). Rabbit serum raised against grape leaf NAD(H)-GDH was kindly provided by Loulakakis and Roubelakis-Angelakis (1990b).…”