Characteristics of Ribulose-1,5-Bisphosphate Carboxylase/ Oxygenase Degradation by Lysates of Mechanically Isolated Chloroplasts from Wheat Leaves1

The lysate from intact chloroplasts mechanically isolated from primary leaves of 9 day old seedlings of wheat (Triticum aestivum L. var Aoba) was incubated in the pH range of 5.5 to 8.5 at 370C for 5 hours. Proteolytic activity against ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) was estimated by disappearance of the large subunit of Rubisco or the appearance of its degradation products. Although the activity in lysates was weak, the products were detected by applying Westem blotting. The degradation products were similar to those obtained when Rubisco was incubated with the lysate of vacuoles isolated from like leaves. Although some of the products were similar to those from vacuole lysates, many were clearly different after incubation of Rubisco with trypsin, V-8 protease, or reactive oxygen (hydroxy radical). Lysates of chloroplasts, pretreated with thermolysin at 40C for 30 minutes, had no proteolytic activity against Rubisco after incubation at 370C for 5 hours. These results show that the proteolytic activity against Rubisco found in lysates of our mechanically isolated chloroplasts was mostly due to the contamination of vacuolar proteases adhering to the outer envelope of the chloroplasts during their isolation. Rubisco2 (EC 4.1.1.39) is responsible for the primary step of CO2 fixation in C3 plants and exists in chloroplasts as a stromal protein. Rubisco content in leaves is proportional to the rate of photosynthesis throughout the life span of leaves. Thus, the degradation of Rubisco in leaves is an important factor in regulating the rate of leaf photosynthesis, but the mechanism of degradation is not known yet. Proteolytic activity against Rubisco has been found in the chloroplasts prepared from the protoplasts ofwheat and barley mature leaves (4, 13). In those studies, the activity was relatively strong and could be assessed by measuring a loss of LSU. There is also a report that mechanically isolated chloroplasts from wheat leaves contained weak proteolytic activity against Rubisco, which could be detected by employing immunoblotting (8). 'This work was supported in part by Grants-in-Aid for Scientific Research (604880051) and for Overseas Scientific Research (63044018) from the Ministry of Education, Science and Culture of Japan. 2 Abbreviations: Rubisco, ribulose1 ,5-bisphosphate carboxylase/ oxygenase; LSU, large subunit of Rubisco; TBS, Tris buffered saline; FCS, fetal calf serum. 1215 We postulated that the proteolytic activity against Rubisco in chloroplast lysates might be due to a contamination of proteases derived from vacuoles during isolation of chloroplasts. This was based on similar pH dependence and similar responses to protease inhibitors (2). The purpose of the research reported in this paper was to clarify whether proteolytic activity against Rubisco exists in chloroplasts in vivo, or is derived from other sources during isolation. The degradation products resulting from incubation of chloroplast lysates with Rubisco were compared to those of proteases from vacuoles, using Western blotting with purified anti-LSU antibody. The possible adhering of vacuolar proteases during isolation was examined by treatment of intact chloroplasts with thermolysin, which eliminated proteins attached to the outer envelope. MATERIALS AND METHODS