The catabolism of ribulose bisphosphate carboxylase from higher plants. A hypothesis

Abstract

Based on current knowledge, a model is proposed for the selective catabolism of ribulose bisphosphate carboxylase (RuBP carboxylase) (EC 4.1.1.39). According to this model, the enzyme is first oxidised and subsequently covalently polymerised. However, enzymatic activities capable of oxidising the enzyme and of preferentially degrading oxidised/polymerised RuBP carboxylase in vivo have not been reported. In this work, we report the existence of both these activities and have developed methodologies capable of measuring their catalytic activities. Detection of the oxidase system involved extraction of the plant tissue incubated under conditions that are known to induce the oxidases ( Lemna minor subjected to osmotic shock and, apparently, all other conditions that affect membrane integrity; Franco et al., Aust. J. Plant Physiol. 19 (1992) 297–307) and incubation under appropriate conditions with purified RuBP carboxylase as the substrate, in the presence of unknown molecular mass factor(s). Oxidation of RuBP carboxylase in vitro occurs via formation of both disulphide and non-disulphide covalent bonds between large subunits (LSUs). Detection of the oxidase system is subsequently achieved by reduced-condition sodium dodecyl sulfide-polyacrylamide gel electrophoresis (R-SDS-PAGE) (by following the accumulation of P65, an intermediate formed by non-disulphide, covalent ligation of one LSU with one small subunit (SSU)) or by anion exchange chromatography (by following the changes in the binding properties of substrate RuBP carboxylase to the fast protein liquid chromatography Mono-Q column). Detection of the proteolytic system involved the previous preparation of 3H-native, 3H-oxidised and 3H-polymerised forms of RuBP carboxylase as substrates for proteolysis. The proteolytic system exhibiting higher affinity towards oxidised and particularly polymerised RuBP carboxylase was extracted from L. minor grown under normal metabolic conditions, L. minor subjected to sulphur starvation and Triticum aestivum leaves deprived of nitrogen. Proteolysis was detected by native PAGE (by running the extract containing the proteases through a native gel containing oxidised RuBP carboxylase embedded in its matrix), R-SDS-PAGE (by following the proteolytic changes in both LSU and SSU) or liquid scintillation counting (by measuring the amount of acid-soluble radioactivity released by the action of the proteases).