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

Abstract

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.