STUDIES ON WHEAT ACETYL COA CARBOXYLASE AND THE CLONING OF A PARTIAL CDNA

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chro matography and electroelution. During purification bovine serum albumi n (BSA) was used to coat Amicon membranes used to concentrate partiall y pure ACCase. Despite further SDS-PAGE/electroelution and microbore H PLC steps BSA remained associated. This presented serious protein sequ encing artefacts which may reflect the affinity of BSA for fatty acids bound to ACCase. To avoid these artefacts the enzyme was digested in gel with Endoproteinase LysC protease without the presence of BSA, and the resulting peptides blotted and sequenced. A partial cDNA (1.85 kb ) encoding ACCase from a wheat embryo library was cloned, which hybrid ised to a 7.5 kb RNA species on northern blot of wheat leaf poly(A)(+) RNA. The partial cDNA therefore represents about 0.25 of the full-len gth cDNA. The clone was authenticated by ACCase peptide sequencing and immune cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequence s (62%). Antibodies raised against wheat acetyl CoA carboxylase were s pecific for a 220 kDa protein from both wheat embryo and leaf. In addi tion, by using a novel quick assay for ACCase that utilised I-125-stre ptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously pu blished molecular mass of 75 kDa allocated to wheat leaf ACCase.