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

• Published in: Plant molecular biology 1994-01, Vol.24 (1), p.21-34
• Main Authors: Elborough, K.M. (Durham Univ. (United Kingdom). Dept. of Biological Sciences), Simon, J.W, Swinhoe, R, Ashton, A.R, Slabas, A.R
• Format: Article
• Language: English
• Subjects: Acetyl-CoA Carboxylase - genetics; Acetyl-CoA Carboxylase - metabolism; ADN; ALBUMINAS; ALBUMINE; ALBUMINS; Amino Acid Sequence; Base Sequence; Biological and medical sciences; Blotting, Northern; Blotting, Western; CEREAL GERMS; CHROMATOGRAPHIE; CHROMATOGRAPHY; CLONACION; CLONAGE; CLONING; Cloning, Molecular; CROMATOGRAFIA; DNA; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; EMBRIONES VEGETALES; EMBRYON VEGETAL; Enzymes; FEUILLE; Fundamental and applied biological sciences. Psychology; GERME DE CEREALE; GERMENES DE CEREALES; HOJAS; LEAVES; LIASAS; LIGASAS; LIGASE; LIGASES; LYASE; LYASES; Metabolism; Molecular Sequence Data; PLANT EMBRYOS; Plant physiology and development; PURIFICACION; PURIFICATION; Sequence Homology, Amino Acid; TRITICUM; Triticum - enzymology; Triticum aestivum
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/bf00040571

Wheat germ acetyl CoA carboxylase (ACCase) was purified by liquid chromatography and electroelution. During purification bovine serum albumin (BSA) was used to coat Amicon membranes used to concentrate partially pure ACCase. Despite further SDS-PAGE/electroelution and microbore HPLC steps BSA remained associated. This presented serious protein sequencing 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 hybridised 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-length cDNA. The clone was authenticated by ACCase peptide sequencing and immuno cross-reactivity of the overexpressed clone. The derived amino acid sequence showed homology with both rat and yeast ACCase sequences (62%). Antibodies raised against wheat acetyl CoA carboxylase were specific for a 220 kDa protein from both wheat embryo and leaf. In addition, by using a novel quick assay for ACCase that utilised 125I-streptavidin, we showed the major biotin containing protein to be 220 kDa in both leaf and germ. This is in marked contrast to the previously published molecular mass of 75 kDa allocated to wheat leaf ACCase.