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716 BIOCHEMICAL SOCIETY TRANSACTIONS expressed/total activity ratio of 74.8%. The expressed/total activity ratio for acini which were not incubated was 5 1.1 %, indicating that dephosphorylation of the enzyme in control acini after 1 h incubation had occurred. Prolactin had no effect on fatty acid synthesis from [I4C]acetatemeasured under the same conditions, whereas insulin increased fatty acid production. The dose-response curve for insulin was again sigmoidal, with approximately 87 munits/ml causing half-maximal effect. At this concentration, the rate of fatty acid synthesis was increased by 25% to 623.4 f 15.5nmol/h per mg of DNA, whereas at an insulin concentration of 53 munits/ml it was hardly affected, 522.8 f 9.6 (nmol/h per mg of DNA) in comparison with control values of 496.8 f 9.1 (nmol/h per mg of DNA). Therefore, around L3 where the changes in enzyme activity were the greatest, the rate of cholesterogenesis in the acini was susceptible to manipulation by changes in the hormonal environment, but at D9, where experimental evidence indicated that the quantity of enzyme was at a minimum, hormone effects were evidently not important. Cholesterol synthesis from acetate was apparently more sensitive to stimulation by insulin treatment than was fatty acid synthesis. Since acetate is a common substrate for both pathways, this result suggests preferential direction of acetate into cholesterol synthesis. R.A.W.S. is supported by an S.E.R.C./C.A.S.E. Postgraduate Studentship. Balasubramanian,S., Goldstein, J. L., Faust, J. R. & Brown, M. S. (1976) Proc. Natl. Acad. Sci. U.S.A. 13, 25642568 Gibbons, G. F., Pullinger, C. R., Munday, M. R. & Williamson, D. H. (1983) Biochem. J . 212, 843-848 Robson, N. A., Clegg, R. A. & Zammit, V. A. (1984) Biochem. J . 217, 143-749 Smith, R. A. W., Middleton, B. & West, D. W. (1985) Biochem. Soc. Trans. 14. 123-124 The basic polypeptide subunit of rape leaf acetyl-CoA carboxylase is a 220 kDa protein A. R. SLABAS, A. HELLYER and H. E. BAMBRIDGE Protein Chemistry Section, Biosciences Division, Unilever Research, Colworth House, Sharnbrook, Bedford, MK44 ILQ, U . K . Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA, which is an essential substrate for fatty acid biosynthesis. The enzyme has been purified from various plant sources and its subunit polypeptide composition has been analysed using polyacrylamide-gel electrophoresis under denaturing conditions. Such studies have not yielded uniform results. We have recently purified the enzyme from maturing seeds of oil seed rape and shown that it is composed of a single high molecular weight 220 kDa polypeptide (Slabas & Hellyer, 1985). The enzyme from wheat germ and parsley suspension culture has also been shown to be composed of a single high molecular weight polypeptide of molecular mass 240 and 210 kDa respectively (Egin-Bulher et al., 1980). Other studies on spinach (Kannangara & Stumpf, 1972, 1973) and other leaf materials (Nikolau et al., 1984) have indicated that the leaf enzyme is composed of one or more low molecular weight polypeptides. There thus appears to be a fundamental difference in the enzyme isolated from seed and leaf material. This in part at least could be due to the relatively lengthy isolation procedure used to purify the enzyme from plant sources. In order to investigate the situation in rape seed we have applied the use of monovalent avidin chromatography to purify the enzyme from rape leaf. Acetyl-CoA carboxylase was purified from rape leaf essentially by our previously decribed procedure (Slabas & Hellyer, 1985). This procedure involves monovalent avidin chromatography as the first step and elution from Blue Separose with ATP as the second step. The preparation was analysed at both stages by SDS/polyacrylamide-gel electrophoresis (Fig. 1). After monovalent avidin chromatography the preparation contained two major bands at 220 kDa and 55 kDa (Fig. 1A). Chromatography on Blue Separose results in a virtually homogeneous preparation of 220 kDa polypeptide. These results demonstrate that, in rape, both the seed and leaf enzymes have a high molecular weight polypeptide and that there is no special low molecular weight leaf form of the enzyme. Egin-Buhler, B., Lloyd, R. & Ebel, J. (1980) Arch. Biochem. Biophys. 203. 9G95 Kannangara, C. G. & Stumpf, P. K. (1972) Arch. Biochem. Biophys. 152, 83-91 Kannangara, C . G. & Stumpf, P. K. (1983) Arch. Biochem. Biophys. 155, 391-399 Nikolau, B. J., Wurtele, E. S. & Stumpf, P. K. (1984) Arch. Biochem. Biophys. 235, 555-561 Slabas, A. R. & Hellyer, A. (1985) Plani Sci. 39, 177-182 A B 97 66 45 Fig. 1. SDS/polyacrylamide-gel electrophoresis of rape leaf acetyl-CoA carboxylase at various stages of purity (A) Monovalent avidin purified. (B) Further purified on Blue Sepharose.The proteins were detected by silver staining and the position of authentic molecular mass markers in kDa is shown on the left-hand side. 1986