Degradation of Ribulose-l,5-bisphosphate Carboxylase/Oxygenase in the Lysates of the Chloroplasts Isolated Mechanically from Wheat (Triticum aestivum L.) Leaves

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Abstract
Intact chloroplasts were isolated mechanically from the primary leaves of 8- to 12-day old seedlings of wheat (Triticum aestivum L.) and purified by Percoll gradient centrifugation. The chloroplasts were lyzed by osmotic shock and the reaction mixtures containing the lysates were incubated in the pH range of 5.3 to 9.4 at 37°C. The degradation of ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39) and its degradation products in the mixtures were examined by using SDS-polyacrylamide gel electrophoresis. RuBisCO-hydrolase activity in the lysates was very weak, and it was difficult to assess the activity by measuring the loss of the amount of the large subunit of RuBisCO on the gels after staining with Coomassie Brilliant Blue. By using immunoblotting method, however, degradation products of RuBisCO could be detected in the reaction mixtures. The hydrolase activity was pronounced in the presence of 0.1 % (w/v) of SDS in the reaction mixtures. Among the products, the 35 kDa fragment was conspicuous and found in the wide range of pHs. This degradation of RuBisCO was inhibited in the presence of leupeptin and N-ethylmaleimide.