Induction of a specific isoenzyme of glutamate dehydrogenase during isolation and the first 48 h of culture of Brassica napus leaf protoplasts

The specific activity of NADH‐glutamate dehydrogenase (GDH, EC 1.4.1.2) in leaf protoplasts ( Brassica napus L. cv. Bronowski) was initially low and progressively increased during culture in Murashige and Skoog (MS) medium and MS (−NH₄) (ammonium nitrate‐free MS) medium in the dark. Native polyacrylamide gel electrophoresis (PAGE) and tetrazolium staining revealed that the high specific activity of NAD‐GDH (deamination) in leaves correlated with the cathodal isoenzymes, and the high specific activity of NADH‐GDH (amination) in leaf protoplasts to the anodal ones. Changes in isoenzyme pattern were correlated with an increase in the specific activity of NADH‐GDH but not with the NADH‐GDH/NAD‐GDH ratio. The increase in NADH‐GDH (amination) activity of leaf protoplasts was correlated with the occurrence of the isoenzyme GDH7, which was not detected in leaves.     

The seven NAD(H)-glutamate dehydrogenase isoenzymes exhibit similar anabolic and catabolic activities

The specific activities of aminating NADH- and deaminating NAD⁺-glutamate dehydrogenase (GDH, EC 1.4.1.2) varied considerably in crude extracts of grapevine ( Vitis vinifera L. cv. Sultanina) callus and were dependent on the nitrogen source of the culture medium. However, dialysis of the enzyme preparations resulted in a significant decrease in the deaminating GDH specific activity while the aminating activity was not affected. The presence of malate in the crude extract resulted in erroneous overestimation of the NAD⁺-GDH activity through the malate dehydrogenase reaction. Thus, in dialysed extracts, the ratio of the NADH-GDH/NAD⁺-GDH specific activities remained relatively constant irrespective of the nitrogen source. In view of this evidence, we now have modified methods for staining both the NADH-GDH and NAD⁺-GDH activities on gels in order to compare the aminating and deaminating activities of each of the 7 GDH isoenzymes. The results from the staining of NADH-GDH and NAD⁺-GDH activity of enzyme preparations from calluses revealed the same isoenzyme profile. Furthermore, separated leaf isoenzymes showed similar activity ratios and kinetic properties. These results may suggest that each one of the 7 isoenzymes have similar in vitro anabolic and catabolic activities.     

Endogen und exogen ausgelöste Änderung des Isoenzymspektrums der NAD-spezifischen Glutamatdehydrogenase im Sproß von Pisum sativum

Summary The isoenzymes of NAD-specific glutamic dehydrogenase (GDH) of Pisum sativum, separated by polyacrylamide gel electrophoresis, constitute two patterns, each of which covers seven individual isoenzymes. One pattern (GDH-I) is found in the cotyledons and young shoots. The second one (GDH-II) occurs together with at least some GDH-I isoenzymes in pea roots. In the shoots of older pea plants GDH-II isoenzymes become visible in addition to the GDH-I pattern.Section of the cotyledons (but not of the roots) of young pea seedlings causes the formation of the complete GDH-II isoenzyme pattern in the shoots within a few hours. It has been verified that the cotyledons specifically suppress the formation of the GDH-II pattern in the young shoot. In older plants which no longer depend on the cotyledons this effect is maintained somewhat less obviously by the root system.In experiments with isolated shoot segments or shoot tips it has been shown that NH ₄ ⁺ reinforces the formation of the GDH-II whereas glucose shows the opposite effect.The formation of the GDH-II isoenzymes in the presence of NH ₄ ⁺ is accompanied by an increase of the specific activity of GDH. Simultaneously the ratio of aminating activity (anabolic reaction) to deaminating activity (catabolic reaction) changes in favor of the anabolic reaction.The results support the supposition that the GDH-I and GDH-II isoenzyme patterns correspond to different molecular forms of one enzyme, the GDH-II representing a form with predominantly anabolic function and the GDH-I a form which has merely metabolic or catabolic function.     

The effect of different carbon and nitrogen sources on the activity of glutamine synthetase and glutamate dehydrogenase in lupine embryonic axes

Embryos of yellow lupine ( Lupinus luteus L. cv. Jantar), deprived of cotyledons, were incubated for 72 h in media containing various combinations of saccharose, ammonia, nitrate, glutamine and asparagine. Induction of glutamine synthetase (GS) was observed in embryos in media containing saccharose while the activity of this enzyme was inhibited by glutamine, asparagine and ammonia in the absence of sugar. The above mentioned nutritional factors had an opposite effect on the activity of glutamate dehydrogenase (GDH). Changes in glutamate dehydrogenase activity were preceded by reverse changes in the activity of glutamine synthetase. The possibility of GDH repression by GS in lupine embryos is discussed.     

Purification and properties of glutamate dehydrogenase in Scots pine (Pinus sylvestris) needles

NADH-dependent glutamate dehydrogenase (GDH. EC 1. 4. 1.2) was isolated from the needles of Scots pine (Pinus sylverstris L.) grown on a rural and on a heavily polluted industrial area, and it was purified about 500 fold. The purification procedure included salt I'ractionation, ion exchange and affinity chromatography. Miehaelis constants for 2-oxoglularale (1.7 mM). for ammonium sultate (19 mM ) and for NADH (42.5 resp. 53 μM) the pH optimum (8.5) the requirements for Ca²⁺ ions, the temperature dependence ofl the enzyme activity (incubation from 0 to 82°C). and the relation between forest region and electrophoretie isoenzyme pattern were determined. The possible role of GDH in the adaptation of plants to ammonia assimilation (detoxification) under stress conditions, particularly with respect to air pollution, is discussed.     

Tobacco isoenzyme 1 of NAD(H)-dependent glutamate dehydrogenase catabolizes glutamate in vivo

Glutamate (Glu) dehydrogenase (GDH, EC 1.4.1.2-1.4.1.4) catalyzes in vitro the reversible amination of 2-oxoglutarate to Glu. The in vivo direction(s) of the GDH reaction in higher plants and hence the role(s) of this enzyme is unclear, a situation confounded by the existence of isoenzymes comprised totally of either GDH beta- (isoenzyme 1) or alpha- (isoenzyme 7) subunits, as well as another five alpha-beta isoenzyme permutations. To clarify the in vivo direction of the reaction catalyzed by GDH isoenzyme 1, [(15)N]Glu was supplied to roots of two independent transgenic tobacco (Nicotiana tabacum) lines with increased isoenzyme 1 levels (S4-H and S49-H). The [(15)N]ammonium (NH(4)(+)) accumulation rate in these lines was elevated approximately 65% compared with a null segregant control line, indicating that isoenzyme 1 catabolizes Glu in roots. Leaf glutamine synthetase (GS) was inhibited with a GS-specific herbicide to quantify any contribution by GDH toward photorespiratory NH(4)(+) reassimilation. Transgenic line S49-H did not show enhanced resistance to the herbicide, indicating that the large pool of isoenzyme 1 in S49-H leaves was unable to compensate for GS and suggesting that isoenzyme 1 does not assimilate NH(4)(+) in vivo.     

Subject index volume 144

Abstract β-xylosidase (EC 3.2.1.37) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50 °C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-d-xylopyranoside as substrate, the K _m and V _max values have been found to be 1.1 mM and 25.6 μmol min⁻¹(mg protein)⁻¹, respectively. Enzyme activity was inhibited by Hg²⁺, Ag²⁺, and Cu²⁺ at a concentration of 1 × 10⁻³ M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the cre A gene product.     

The involvement of glutamate dehydrogenase in the adaptation of mitochondria to oxidize glutamate in sucrose starved pea embryos

Embryos of pea (Pisum sativum L. cv Sol) deprived of cotyledons were cultured for 3 days in medium with or without sucrose. Respiratory activity of embryos (intact) as well as the ability to oxidize glutamate by mitochondria isolated from embryos were studied. Respiration of intact embryos grown in sucrose supplemented medium was more intensive than in the starved ones. Transfer of the starved embryos to the sucrose-containing medium induced the increase in the intensity of O₂ consumption. Mitochondria isolated from both starved and control embryos exhibited respiratory control. Mitochondria isolated from embryos cultured in the absence of sucrose showed higher (about 60 %) ability to oxidize glutamate and α-ketoglutarate than mitochondria from embryos grown in sucrose containing medium. The absence of sucrose in the medium led to a rapid increase in the specific activity of glutamate dehydrogenase (NADH-GDH and NAD-GDH) and it was accompanied by changes in izoenzymatic pattern of enzyme. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase may be responsible for the increase of glutamate oxidation by mitochondria of pea embryos. Electrophoretic separation of glutamate dehydrogenase isolated from embryos cultured in medium without sucrose showed the presence of ca. 17 isoenzymes while in non-starved embryos only 7 isoenzymes were identified. However, the addition of sucrose to starved embryos after 24 hours of cultivation led to a decrease in glutamate dehydrogenase activity (up to 40 %) but it did not cause the changes in isoenzymatic pattern. These results suggest that in the conditions of sucrose starvation glutamate dehydrogenase maybe responsible for the increase of glutamate oxidation by mitochondria of pea embryos. The posibility of glutamate dehydrogenase regulation by sucrose is discussed.     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

A role for chromosomal protein HMGN1 in corneal maturation

Abstract Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1 ^−/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of suprabasal wing cells, and its most superficial cell layer blisters. In mature Hmgn1 ^−/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1 ^−/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1 ^−/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.     

NADP+-dependent glutamate dehydrogenase from the obligate methylotroph methylobacillus flagellatum

Abstract NADP-dependent glutamate dehydrogenase (GDH; E.C.1.4.1.4) was purified from an obligate methylotroph Methylobacillus flagellatum using ammonium sulphate precipitation, DEAE-Sepharose and dye-ligand Procion red HE3B column chromatography and Sephacryl S-200 gel-filtration. The M_r of the native enzyme was estimated to be 300 000 (±5000). The enzyme consists of six identical subunits with an M_{r of} 47 000 (±3000) (SDS-PAGE). The enzyme has a pH optimum of 8.0 when participating in amination and 9.5 in deamination. Michaelis-Menten kinetics were observed for both reactions. The apparent K_m values were 1.33 mM, 0.032 mM, 11.5 mM, 7.0 mM and 0.014 mM for α-ketoglutarate, NADPH, NH₄⁺, glutamate and NADP⁺, respectively. The enzyme was highly specific for all the substrates and was insensitive to inhibitors. It plays an exclusively anabolic role in the cells.     

Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio

The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, α and β, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of α- and β-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of β-polypeptide and of the more cathodal isoenzymes, whereas α-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the α- and the catabolic reaction with the β-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions.     

Organspezifische multiple Formen der Glutamatdehydrogenase in Medicago sativa

Summary The alteration of the multiple forms of NAD-dependent glutamic dehydrogenase (GDH) during the development of Medicago sativa is investigated by means of polyacrylamide electrophoresis. Seed germination is accompanied by a characteristic change of the GDH-isoenzyme pattern. Seeds contain seven isoenzymes, which gradually decrease in number during germination. At the same time a pattern of new isoenzymes becomes visible. The seed pattern is called GDH-I and the later appearing pattern GDH-II. GDH-I is characteristic for the cotyledons, whereas GDH-II is the typical pattern of the root system. Shoots produce a mixed pattern composed of the GDH-II isoenzymes as well as some GDH-I isoenzymes.These isoenzyme patterns are organ specific. No qualitative change occurs during further development of the plants and during growth in the presence of different inorganic and organic N-sources in the culture medium.All the individual isoenzymes are found predominantly in the particulate fraction. They represent stable forms which are not altered by variation of the conditions of enzyme extraction or during enzyme purification. Re-electrophoresis of the individual isoenzymes following elution from the polyacrylamide gels reveals only one specific band. The molecular weights of all the distinctive isoenzymes are identical.There is some evidence that the different isoenzymes represent conformational forms of one enzyme, and it is postulated that the GDH-I isoenzymes are correlated to a normal metabolic (or catabolic) function of the enzyme, whereas the GDH-II isoenzymes are responsible for a primarily anabolic function of glutamic dehydrogenase.     

An extracellular β-galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall-polysaccharide hydrolysis

β-Galactosidase (β-Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β-Galase (β-Galase-II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM-Sephadex C-50. DEAE-Sepharose CL-6B and Sephacryl S-200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S-200HR gel-permeation, and 60 kDa by SDS-PAGE after treatment with SDS and 2-mercaptoethanol. The pI was 6.5. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyranoside were 0.17 m M and 31.9 μmol (mg protein)^-1, h^-1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co²⁴, Cu²⁺, Hg^2-, p -chloromercuribenzoate (PCMB) and D-galactono-1,4-lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo-fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine