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

Based on current knowledge, a model is proposed for the selective catabolis m of ribulose bisphosphate carboxylase (RuBP carboxylase) (EC 4.1.1.39). Ac cording to this model, the enzyme is first oxidised and subsequently covale ntly polymerised. However, enzymatic activities capable of oxidising the en zyme and of preferentially degrading oxidised/polymerised RuBP carboxylase in vivo have not been reported. In this work, we report the existence of bo th these activities and have developed methodologies capable of measuring t heir catalytic activities. Detection of the oxidase system involved extract ion 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; France et al., Aust. J. Pl ant Physiol. 19 (1992) 297-307) and incubation under appropriate conditions with purified RuBP carboxylase as the substrate, in the presence of unknow n molecular mass factor(s). Oxidation of RuBP carboxylase in vitro occurs v ia formation of both disulphide and non-disulphide covalent bonds between l arge subunits (LSUs). Detection of the oxidase system is subsequently achie ved by reduced-condition sodium dodecyl sulfide-polyacrylamide gel electrop horesis (R-SDS-PAGE) (by following the accumulation of P65, an intermediate formed by non-disulphide, covalent ligation of one LSU with one small subu nit (SSU)) or by anion exchange chromatography (by following the changes in the binding properties of substrate RuBP carboxylase to the fast protein l iquid chromatography Mono-Q column). Detection of the proteolytic system in volved the previous preparation of H-3-native, H-3-oxidised and H-3-polymer ised forms of RuBP carboxylase as substrates for proteolysis. The proteolyt ic system exhibiting higher affinity towards oxidised and particularly poly merised RuBP carboxylase was extracted from L. minor grown under normal met abolic conditions, L. minor subjected to sulphur starvation and Triticum ae stivum leaves deprived of nitrogen. Proteolysis was detected by native PAGE (by running the extract containing the proteases through a native gel cont aining oxidised RuBP carboxylase embedded in its matrix), R-SDS-PAGE (by fo llowing the proteolytic changes in both LSU and SSU) or liquid scintillatio n counting (by measuring the amount of acid-soluble radioactivity released by the action of the proteases). (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.