The properties of the large subunit of maize ribulose bisphosphate carboxylase/oxygenase synthesised in Escherichia coli.
The maize chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase has been expressed in Escherichia coli in vivo. This enables the properties of the native large-subunit polypeptide to be examined in the absence of small-subunit polypeptides, and avoids the use of denaturing agents. The product synthesised in bacteria is slightly larger (Mr 54300) than the form present in the chloroplast (Mr 53 300), suggesting the involvement of a precursor polypeptide. In addition several smaller polypeptides are synthesised, predominantly of molecular mass 41 and 30 kDa, but also some of 44 and 12-14 kDa. Pulse-chase experiments with [35S]methionine indicate that all the immunoprecipitable polypeptides are stable. The smaller products are probably the result of premature termination of translation. Virtually all of the large subunits are insoluble, whether synthesised at levels of 100-200 molecules per cell, or up to 60 000 molecules per cell. A small amount of the full-length polypeptide is soluble, but the major soluble product, as determined by sucrose gradient centrifugation, is a polypeptide of molecular mass 12-14 kDa. Ribulose bisphosphate carboxylase activity was undetectable in cell extracts, and binding of a mixture of the radiolabelled transition state analogues carboxyribitol 1,5-bisphosphate and carboxyarabinitol 1,5-bisphosphate could not be detected. It is proposed that other components are required to prevent the large subunit from adopting an inactive, insoluble conformation after, or during, synthesis.
D002384:Catalysis; D002499:Centrifugation, Density Gradient; D002736:Chloroplasts / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D008970:Molecular Weight; D010105:Oxygenases / Q000096:biosynthesis*; D010446:Peptide Fragments / Q000096:biosynthesis; D012273:Ribulose-Bisphosphate Carboxylase / Q000096:biosynthesis*; D012995:Solubility; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D010446:Peptide Fragments; D010105:Oxygenases; D012273:Ribulose-Bisphosphate Carboxylase