Studies on wheat acetyl CoA carboxylase and the cloning of a partial cDNA

摘要: Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) used to coat Amicon membranes concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution microbore HPLC steps BSA remained associated. This presented serious protein sequencing artefacts which may reflect the affinity of for fatty acids bound To avoid these enzyme digested in gel with Endoproteinase LysC protease without presence BSA, resulting peptides blotted sequenced. A partial cDNA (1.85 kb) encoding ACCase from a wheat embryo library cloned, hybridised 7.5 kb RNA species on northern blot leaf poly(A)+ RNA. The therefore represents about 0.25 full-length cDNA. clone authenticated peptide immuno cross-reactivity overexpressed clone. derived amino acid sequence showed homology both rat yeast sequences (62%). Antibodies raised against were specific 220 kDa leaf. In addition, using novel quick assay that utilised 125I-streptavidin, we major biotin containing be germ. is marked contrast previously published molecular mass 75 allocated