Cell-free synthesis of active ribulose-1,5-bisphosphate carboxylase in the mesophyll chloroplasts of Sorghum vulgare.
Chloroplast and whole leaf cell RNA from Vigna sinensis, a C3 plant were used as exogenous templates for translation in a cell-free light-dependent system of isolated chloroplasts from Sorghum vulgare, a C4-type plant. Analysis of immunoprecipitates of the translated products with the total cellular RNA on sodium dodecyl sulfate polyacrylamide gels revealed the synthesis of both the subunits of ribulose-1,5-bisphosphate carboxylase. Similar analysis of the product translated with the RNA from Vigna chloroplasts, indicated the synthesis of only the large subunit of the carboxylase. Apparently the chloroplast protein synthetic machinery is capable of translating the mRNA for the smaller subunit of this protein as well, which is known to be translated in the cytoplasmic ribosomal system. Sufficient quantities of ribulose-1,5-bisphosphate carboxylase were synthesised in vitro in the preincubated chloroplast system with the whole cell RNA from the C3 plant to assay the ribulose 1,5-bisphosphate-dependent carboxylation. The newly synthesised protein in the cell-free system is identical in many ways to the native enzyme including the Mg2+ concentration-dependent shift in pH optima towards neutral side. It is specifically inhibited by anti-native ribulose-1,5-bisphosphate carboxylase and pyridoxal 5'-phosphate.
D002262:Carboxy-Lyases / Q000096:biosynthesis*; D002474:Cell-Free System; D002736:Chloroplasts / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010944:Plants / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000378:metabolism; D013698:Templates, Genetic
D012313:RNA; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D011188:Potassium