Co-purification,co-imniunoprecipitation,and coordinate expression of acetyl-coenzyme A carboxylase activity,biotin carboxylase,and biotin carboxyl carrier protein of higher plants

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a regulatory enzyme of fatty acid synthesis, and in some higher-plant plastids is a multi-subunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxyl transferase (CT). We recently described a Nicotiana tabacum L. (tobacco) cDNA with a deduced amino acid sequence similar to that of prokaryotic BC. We here provide further biochemical and immunological evidence that this higher-plant polypeptide is an authentic BC component of ACCase. The BC protein co-purified with ACCase activity and with BCCP during gel permeation chromatography of Pisum sativum L. (pea) chloroplast proteins. Antibodies to the Ricinus communis L. (castor) BC co-precipitated ACCase activity and BCCP. During castor seed development, ACCase activity and the levels of BC and BCCP increased and subsequently decreased in parallel, indicating their coordinate regulation. The BC protein comprised about 0.8% of the soluble protein in developing castor seed, and less than 0.05% of the protein in young leaf or root. Polypeptides cross-reacting with antibodies to castor BC were detected in several dicotyledons and in the monocotyledons Hemerocallis fulva L. (day lily), Iris L., and Allium cepa L. (onion), but not in the Gramineae species Hordeum vulgare L. (barley) and Panicum virgatum L. (switchgrass). The castor endosperm and pea chloroplast ACCases were not significantly inhibited by long-chain acyl-acyl carrier protein, free fatty acids or acyl carrier protein. The BC polypeptide was detected throughout Brassica napus L. (rapeseed) embryo development, in contrast to the multi-functional ACCase isoenzyme which was only detected early in development. These results firmly establish the identity of the BC polypeptide in plants and provide insight into the structure, regulation and roles of higherplant ACCases.Abbreviations ACCase acetyl-CoA carboxylase - ACP acyl carrier protein - BC biotin carboxylase - BCCP biotin carboxyl carrier protein - CT carboxyl transferase - MF multi-functional - MS multi-subunit We thank our colleagues Nicki Engeseth and Vicki Eccleston for advice on fatty acid analysis and Sarah Hunter for providing the developing Iris seed. This work was supported in part by grant MCB 9406466 from NSF. Acknowledgement is also made to the Michigan Agriculture Experiment Station for its support of this research.