Download The basic polypeptide subunit of rape leaf acetyl

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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