The characterization of senescence-associated endopeptidase (EP) isoenzymes in wheat (Triticum aestivum L. cv. Yangmai 158) leaves during dark-induced senescence was performed. It was found that there was much higher endoproteolytic activity in dark-induced wheat leaves than in control. Six endopeptidase isoenzymes (EP1-EP6) were identified by natural gradient-polyacrylamide gel electrophoresis (PAGE) co-polymerized gelatin in the gel, five of which (EP1, EP2, EP4, EP5 and EP6) were only detected in senescing leaves. Treatment with 6-benzyl aminopurine (6-BA) delayed the expression of these EP isoenzymes and abscisic acid (ABA) accelerated it. The activity of EP3 could be detected at a wider range of pH and temperature levels while EP4, EP5 and EP 6 could be only detected at pH 4-5 and 30 -45 degreesC, EP1 and EP2 at pH 3-5 and 30-45 degreesC. All of the EP isoenzymes showed high thermal stability, especially EP3, EP5 and EP6 which still had activitiy even by incubation at 55 degreesC for 1 h. By using different class-specific inhibitors, EP1 and EP2 were characterized as metal-dependent cysteine-proteases, EP4 as a serine-protease.
