Teresa Lehmann

Stress-induced changes in glutamate dehydrogenase activity imply its role in adaptation to C and N metabolism in lupine embryos.

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity and isoenzyme pattern was investigated in isolated embryos of lupine (Lupinus luteus L.), cultured in vitro in a medium with sucrose (+S) or without sucrose (-S) and exposed to cadmium (Cd) and lead (Pb) stress. Sucrose starvation of lupine embryos led to a rapid increase in the specific activity of GDH, immunoreactive beta-polypeptide and it was accompanied by appearance of new cathodal isoforms of enzyme. This suggests that isoenzymes induced in lupine embryos by sucrose starvation combine into GDH hexamers with the predominance of beta-GDH subunits synthetized under GDH1 gene control. The addition of sucrose to the medium caused an opposite effect. Along with upregulation of catabolic activity of GDH by sucrose starvation, activity of proteolytic enzymes was also induced. These data can point to regulatory mechanism implying a sucrose dependent repression of the GDH1 gene according to the mechanism of catabolic repression. Treatment of embryos with Cd(2+) or Pb(2+) resulted in ammonium accumulation in the tissues, accompanied by an increase in anabolic activity of GDH and activity of anodal isoenzymes, in both (+S) and (-S) embryos without new de novo synthesis of alpha subunit proteins. Thus, GDH isoenzyme profiles may reflect the physiological function of GDH, which appears to be an important link of metabolic adaptation in cells, aimed at using carbon sources other than sugar during carbohydrate starvation (catabolic activity of GDH) and protecting plant tissues against ammonium accumulated because of heavy metal stress (anabolic activity of GDH).

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.

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity in lupine embryos treated with inhibitors of RNA and protein synthesis

The modifying effect of sucrose on GDH activity and isoenzyme pattern in isolated embryos of lupine subjected to treatments with inhibitors of RNA synthesis (transcription inhibitors: actinomycin D and cordycepin) and protein synthesis (cycloheximide and chloramphenicol) was investigated. Sucrose starvation of embryos caused the increase of total activity of GDH(NADH-GDH and NAD-GDH) more than twice. Supply of sucrose to sucrose starved embryos caused a reduction of enzyme activity by 40 %. Electrophoretic analysis showed the presence of ca 17 isoenzymes of glutamate dehydrogenase in embryos grown for 72 h in medium with sucrose, while sucrose starvation increased the number of isoenzymes up to 22 forms. Addition of sucrose to sucrose starved embryos after 24 h of cultivation caused the inhibition of synthesis of new isoenzymes. This down-regulation by sucrose was blocked when sucrose was added together with cycloheximide, CHX, (0.025 mM). Treatment of sucrose fed and sucrose starved embryos with cycloheximide (0.020 mM) inhibited protein synthesis by 58 % and 24 %, respectively. The addition of cycloheximide (0.025 mM) to sucrose starved embryos decreased by 60 % NADH-GDH and by 30 % NAD-GDH activity and reduced the spectrum of isoenzymes. CHX treatment did not lead to a significant reduction of enzyme activity and isoenzyme pattern in sucrose fed embryos. The chloramphenicol (CMP) treatment (1 mM) stimulated the total GDH activity, 2.5 fold and 1.5 fold, in sucrose fed and sucrose starved embryos, respectively. Addition of CMP (10 mM) to the media did not affect GDH activity. Both concentrations of CMP caused no significant changes in the isoenzymatic pattern of enzyme in sucrose starved embryos, but induction of new isoenzymes was observed in sucrose fed embryos treated with CMP. In the case of using RNA synthesis inhibitors only cordycepin inhibited the total GDH activity (by ca 25 %) but neither actinomycin D or cordycepin caused any changes in isoenzyme pattern of GDH. The possible mechanism of sugar-mediated regulation of GDH activity is discussed.

Metabolism of amino acids in germinating yellow lupin seeds. II. Pathway of conversion of aspartate to alanine during the imbibition

The incorporation of 14C-aspartate during the imbibition of yellow lupin seeds resulted in the production of 14C-alanine and 14CO2. On the basis of tracer and enzymatic assays, conducted in vitro on the extract obtained from lupin seeds, it is postulated that aspartate can be converted to oxaloacetate, then, by phosphoenolopyruvate and pyruvate to alanine. This pathway can be catalyzed by the following enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, pyruvate kinase and alanine aminotransferase.

Changes in the activity of phosphoenolpyruvate carboxylating enzymes in germinating yellow lupin seeds

The rate of phosphoenolpyruvate carboxylation by extracts from germinating lupin seeds was measured through the H14CO3 fixation. PEP carboxylation in seed axes increased during their imbibition, mainly as a result of the increase in the activity of PEP carboxylase [EC 4.1.1.31]. However, the activity of PEP carboxykinase [EC 4.1.1.38], present during the first 3 hours of imbibition, as well as the activity of PEP-carboxykinase [EC 4.1.1.49], after 24 hours of imbibition, have also been shown. Possible physiological role of the changes in the activity of PEP carboxylases during lupin seeds germination is discussed.