Diurnal Changes in Asparaginase Activity in Pea Leaves: II. REGULATION OF ACTIVITY

Pea leaf asparaginase is stabilized by asparagine and aspartateduring incubation. In crude extracts this effect was enhancedby products of the light reaction (NADPH, NADH, or reduced ferredoxin),but these compounds were ineffective on the purified enzyme,or in the absence of asparagine. MgATP, MgADP and oxidized ferredoxinreduced asparaginase activity in purified preparation reducedor oxidized glutathione had no effect. Asparaginase activitydoes not appear to be modulated via phosphorylation/dephosphorylation.The presence of calcium during extraction increased asparaginaseactivity more than 2-fold, but addition of calcium to extractsprepared in its absence had no effect; calmodulin had no effecton activity. Co-extraction of light- and dark-treated tissueshowed that soluble factors are not responsible for the diurnalvariation in asparaginase activity. Association of asparaginasewith membranes did not account for changes in extractable activity.Use of the protein synthesis inhibitors cycloheximide, puromycin,emetine, actinomycin D and cordycepin and the thiol proteaseinhibitor leupeptin suggested that mRNA and protein synthesisare required for the increase of asparaginase activity duringthe light period and that proteolytic degradation accounts forthe decrease during the dark. Key words: Pisum sativum, asparaginase, protein synthesis, proteolysis.     

Diurnal Changes in Asparaginase Activity in Pea Leaves: I. THE REQUIREMENT FOR LIGHT FOR INCREASED ACTIVITY

During the growth of leaves of Pisum sativum L., levels of asparaginase(E.C. 3.5.1.1 [EC] ) showed a diurnal variation during a 3 d periodof leaf expansion, increasing in the light and decreasing inthe dark period; the greatest diurnal variation being foundin half-expanded leaves. Asparaginase activity in half-expandedleaves reached a maximum after 4 h exposure to light and thisactivity was maintained over the rest of the light period. Changesin asparaginase activity were not influenced by diurnal temperaturechanges. The increase in asparaginase activity during the lightperiod was directly proportional to the photon flux densityover the range 0–285 µmol m^-2 s^-1 PAR. The increaseof asparaginase activity during illumination of detached leaveswas inhibited by the photosynthetic electron transport inhibitors3-(3', 4'-dichlorophenyl)-1, 1-dimethylurea (DCMU) and atrazine.These observations indicate that the increase in asparaginaseactivity in half-expanded leaves is dependent upon non-cyclicelectron transport. Key words: Pisum sativum, asparaginase, photosynthetic electron transport