Assay and Comparative Characterization of the Proteolytic Degradation of Isolated Small Subunit and Holoenzyme of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chloroplasts from Rye Leaves

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Summary The cleavage of purified and fluorescent or radioactive labeled ribulose-1,5-bisphosphate carboxylase/oxygenase and its isolated small subunit by proteolytic activities of purified chloroplasts from rye leaves ( Secale cereale L.) were compared. Degradation of the small subunit was mainly catalysed by the soluble supernatant fraction of chloroplasts or ribosome-deficient plastids, with highest activity at pH 6. Proteolytic cleavage of the ribulose-1,5-bisphosphate carboxylase holoenzyme was found in the soluble supernatant, but also to some extent associated with the membrane fraction of chloroplasts, having its maximal activity at pH 8. The K m for cleavage of the holoenzyme was 1.2 μM; the K m for cleavage of the small subunit was 52.7 μM. Proteolytic breakdown of the small subunit was enhanced by the addition of SDS, particularly in the presence of dithioerythritol. The activities of the proteolytic degradation of labeled ribulose-1,5-bisphosphate carboxylase/oxygenase and its small subunit were not specifically affected by various protease inhibitors that were representative for the different mechanistic classes of proteases. Proteolytic activities were enriched to higher specific activities by affinity chromatography with activated Thiol-Sepharose 4B. A function of a small-subunit-specific protease in chloroplast biogenesis for the control of the subunit stoichiometry of the ribulose-1,5-bisphosphate carboxylase/oxygenase is discussed.