| The changes and characterization of endopeptidase (EP) isozymes and the degradation of Rubisco were studied during wheat (Triticum aestivum L. cv.Yanmai 158) leafsenescence.1, The changes of endopeptidase isozymes in wheat leaf senescenceThe changes in number and activity of endopeptidase isozyme in wheat leaves were studied during natural and dark-induced senescence. The total activity of EPs increased at first and then decreased during both senescences. The changes of endopeptidase (EP) isozymes in wheat leaf senescence were studied by using natural gradient-polyacrylamide gel electrophoresis (PAGE) co-polymerized with hemoglobin or gelatin in the gel. Four emerging EP isozymes were detected in dark-induced senescence with hemoglobin in the gel, and five during natural senescence. Of all the EP isozymes, different isoenzymes appeared at the different stage of senescence, and the activity of some originally existed EP isozymes increased. When with gelatin co-polymerized in the gel, six senescence -associated EPs were detected in senescing leaves, of which five were induced by senescence. Of all the EPs, EP3 existed in young leaves but its activity increased during senescence. The EP isozymes pattern with gelatin as subtract was similar to the pattern with Rubisco as subtract. While using SDS-gelatin-PAGE, only one emerging EP isoenzyme was detected. It implied that most of the EPs expressed during senescence were sensitive to SDS or these proteases were composed of multisubunits. It was observed that the patterns of EP isozymes during natural senescences and dark-induced senescence were similar. So dark-induced senescence could be used as a pattern to study the characters of the protease in natural senescing leaves.2,Characterization of senescence-associated endopeptidase iso-zymes in wheat leavesThe characterization of senescence-associated endopeptidase isozymes in wheat leaves during dark-induced senescence was performed. Six senescence-associated endopeptidase isoenzymes were identified by using natural gradient-polyacrylamide gel electrophoresis (PAGE) co-polymerized with gelatin in the gel, five of which(EPl, EP2, EP4, EP5 and EP6) were only detected in senescing leaves. Actidione, the inhibitor of 80S ribosome, could inhibit the expression of these senescenc-associated EPs, but chloro-mycetin, the inhibitor of 60S ribosome, had no effect. Treatment with 6-BA delayed the expression of these EP isoenzymes, but ABA accelerated it. The activity of EP3 could be detected at a wider range of pH values and temperature levels, while EP4, EP5 and EP 6 could be only detected at pH 4-5 and 30C-45C, EP1 and EP2 at pH 3-5 and 30C-45C. All of the EP isoenzymes showed high thermal stability, especially EP3 ,EP5 and EP6, which still had activities even by incubation at 55C for 1 hour. By using different class-specific inhibitors, EP1 and EP2 were characterized as metal-dependent cysteine -proteases and EP4 as a serine-protease. Additionally, EP5 and EP6 could be partially inhibited by serine-protease inhibitors, but all of the inhibitors used had no effects on EP3.3 , The degradation of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) during wheat leaf senescenceThe degradtion of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence and natural senescence was studied. An in vivo degradation product of Rubisco large subunit (LSU) with molecular weight of 50kD was detected by SDS-PAGE and immunoblotting with antibody against tobacco Rubisco in dark-induced senescence and natural senescence. It was the first time that the Rubisco in vivo degradation product was detected. The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53kD to 50kD. And LSU could be fragmented to 50kD at 30-35C and at pH5.5 in crude enzyme extracts of wheat leaves dark-induced for 0h, but in the extracts of wheat leaves dark-induced for 48h, the frag...
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