Proteolysis of Endogenous Substrates in Senescing Oat Leaves

Proteolysis of ribulose bisphosphate carboxylase (RuBPCase) during senescence was monitored using oat leaf segments (Avena sativa cv Victory), kept in the dark. We here report the development of a novel approach for measuring protein degradation of endogenous substrates both in situ and in vitro in crude extracts using specific antibodies against highly purified polypeptides. The proteolytic products were separated on sodium dodecyl sulfate-gels. They were then electrotransferred onto nitrocellulose paper and identified with specific antibodies to both the large and small subunits of RuBPCase. We could show differences in pH optima between two proteases degrading the subunits of RuBPCase. While both subunits were best hydrolyzed in acid and basic pH, they degraded differently at neutral pH. Furthermore, the large subunit displayed a different pattern of degradative products at the different pH levels. Older leaf segments, which were incubated in darkness, underwent enhanced proteolysis, as compared with young ones. These results show the advantages of the assay in demonstrating: (a) in situ proteolysis of specific substrates in crude extracts without further purification; (b) in vitro differential proteolysis of endogenous substrates during senescence.