Download Purification and expression of an Abelson-murine-leukaemia

277
604th MEETING, CAMBRIDGE
toxic elemental mercury is released from the enzyme, and
diffuses out of the cell. The unidentified gene products at coordinates 0.1 17-0.150 and 0.363-0.552 may be involved in
transport of Hg(I1) from the membrane to the mercuric reductase, Or may be
in the transport Of Hg(o) Out Of
the cell.
Brown, N. L., Ford, S. J., Pridmore, R. D. & Fritzinger, D. C.
(1983) Biochemistry 22, 40894093
Foster, T. J., Nakahara, H., Weiss, A. A. & Silver, S. (1979) J .
Bacrerioi.
167-181
Fox, B. S. & Walsh, C. T. (1982) J. Biol. Chem. 257, 2498-2503
Fox, B. S, & Walsh, C. T. (1983) Biochemistry 22. 40824088
Cell-free translation of the uncoupling protein of brown-fat mitochondria
FRfiDfiRIC BOUILLAUD,* DANIEL RICQUIER*
and JEAN THIBAULTt
*Luboratoire de Physiologie Comparhe (CNRS-L.A. 307).
UniversitP Pierre et Marie Curie, 4 Place Jussieu, F-75230
Paris Cedex 05, and tLuboratoire de Biochimie Cellulaire
du Collgge de France, F-75231 Paris Cedex 05, France
The thermogenic uncoupling of brown-fat mitochondria is
related to the activity of a proton channel through the inner
membrane (Nicholls, 1979); the activity of this channel is
associated with the presence of a characteristic 32000-M,
uncoupling protein in the membrane (Heaton et al., 1978).
The amount of this component is greatly increased when the
thermogenic activity of the tissue is stimulated, either during cold adaptation (Ricquier & Kader, 1976; Ricquier et
al., 1979) or after development of a pheochromocytoma
tumour (Ricquier et al., 1983a).
Most studies on biogenesis of mitochondria1 proteins
have been conducted with yeast, and they concern proteins
which are common to all types of mitochondria (reviewed
by Neupert & Schatz, 1981). We studied the synthesis in
vitro of the uncoupling protein of brown-fat mitochondria,
which is probably a specific component of these mitochondria. The aims of this study were to measure the size of
the synthesized protein and to try to characterize the corresponding mRNA.
mRNA was prepared from the brown fat of three groups
of rats: control animals kept at room temperature, rats exposed at 5°C for 7 days and rats bearing a pheochromocytoma tumour. The synthesis was carried out in a rabbit
cell-free system digested by Micrococcus nuclease (Pelham
& Jackson, 1976).
Analysis of newly synthesized proteins showed, among
several differences, a significantly increased labelling of a
32000-Mr band in synthesis directed by m R N A from
thermogenic tissues. Immunoprecipitation with antibodies
raised against the uncoupling protein (Ricquier et al.,
19836) demonstrated that this component was synthesized
with the same apparent M , as the mature form (Ricquier et
al., 1983~).It was also concluded that cold exposure and
pheochromocytoma induced an increase in the amount of
specific mRNA coding for the uncoupling protein. The size
of the mRNA was determined and found to be roughly equivalent to 1.7 kilobases, which means that the m R N A could
contain 1000 non-coding nucleotides. Experiments using recombinant complementary D N A technology should yield
further information.
Financial support from C.N.R.S. is acknowledged.
Heaton, G. M., Wagenvoord, R. J., Kemp, A. & Nicholls, D. G .
(1978) Eur. J. Biochem. 82, 515-521
Neupert, W. & Schatz, G . (1981) Trends Biochem. Sci. 6, 1 4
Nicholls, D. G. (1979) Biochim. Biophys. Acta 549, 1-29
Pelham, H. R. B. &Jackson, R. J. (1976) Eur. J . Biochem. 67,247256
Ricquier, D. &Kader, J. C. (1976) Biochem. Biophys. Res. Commun.
73, 577-583
Ricquier, D., Mow, G. & Hemon, Ph. (1979) Can. J. Biochem. 57,
1262-1 266
Ricquier, D., Mory, G., Nechal, M. & Thibault, J. (1983~)Am. J.
Physiol. 245, C 172-C 177
Ricquier, D., Barlet, J. P., Garel, J.-M., Combes-George, M. &
Dubois, M.-P. (19836) Biochem. J. 210, 859-866
Ricquier, D., Thibault, J., Bouillaud, F. & Kuster, Y. (1983~)J.
Biol. Chem. 258. 6675-6677
Purification and expression of an Abelson-murine-leukaemia-virus-encodedprotein kinase
from Escherichia coli
J. GORDON FOULKES, JEAN Y. J. WANG,
NANCY C. ANDREWS and DAVID BALTIMORE
Centerfor Cancer Research, Whitehead Institute for
Biomedical Research, Massachusetts Institute of Technology,
Cambridge. M A 02139, U.S.A.
Abelson murine leukaemia virus (A-MuL virus) is a
member of a replication-defective rapidly transforming
group of retroviruses. A-MuL virus can induce leukaemia in
uivo, as well as transform bone-marrow cells and some
established mouse cell lines in vitro (Risser, 1982). The
ability to transform cells has been attributed to the single
protein encoded by the viral genome. The only known activity of this protein is to act as a tyrosyl-protein kinase (Witte
et al., 1980; Wang et al., 1982). Although phosphotyrosine
concentrations are elevated in cells transformed by A-MuL
virus (Sefton et al., 1981), the function of such phosphoproteins remains to be established. To gain further insight into
this system, we decided to transfer the coding sequences for
VOl. 12
this enzyme from the viral genome on to a vector that would
allow expression in Escherichia coli. Vector pCS4 contains
the P, promoter of I bacteriophage, a ribosome-binding sequence and an ATG codon, followed by 0.24 kilo-base-pairs
of sequences coding for small t protein of Simian virus
SV40. A temperature-sensitive c 1 gene of I bacteriophage is
also present, so that transcription from the PRpromoter is
repressed at 30°C and induced at 42°C (Wang et al., 1982).
To this vector we added 1.2 kilo-base-pairs of sequences
coding for a specific N-terminal region of the A-MuL-virusencoded kinase. This construct should therefore produce a
fusion protein consisting of 80 amino acid residues of the
small t protein and 404 amino acid residues of the kinase. In
the present communication we report the purification of
this protein to homogeneity. The first step was to develop a
specific assay for the enzyme, namely the transfer of the yphosphate group of [y-3ZP]ATPto the single tyrosine residue
of the octapeptide angiotensin 11. Bacteria, containing the
vector, were grown at 30°C in 9.0 litres of L broth, enriched
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BIOCHEMICAL SOCIETY TRANSACTIONS
with glucose (20g/l) and a mineral-salts media, until an absorbance of A660 = 5.0 was reached. The culture was then
shifted to 42"C, to induce synthesis of the kinase and incubated for 4h (final absorbance of A660 = 12.0). Bacteria
were then harvested by centrifugation, divided into four
55g batches and stored at -70°C until processed. The
batches were thawed on ice, the bacteria were lysed by sonication, and the preparation was then clarified by centrifugation. Kinase activity, located in the soluble fraction, was
fractionated by batchwise chromatography on DEAEcellulose, hydroxyapatite and Sephacryl S200. Kinase activity was further purified by chromatography on Affi-Gel
Blue and high-pressure liquid chromatography. The AMuL-virus-encoded kinase autophosphorylates on tyrosine
residues. These phosphotyrosine residues do not appear to
undergo turnover in E. coli. The final purification step
therefore employs a monoclonal antibody, directed towards
phosphotyrosine, to bind the kinase, which is then eluted by
the addition of phenyl phosphate. The overall purification
of the A-MuL-virus-encoded kinase is 5000-fold, with a 1015% yield, and can be accomplished in 2-3 days. A 30pg
yield of purified enzyme can therefore be obtained from 50g
of E. coli.
Risser, R. (1982) Biochim. Biophys. Actu 651, 213-244
Sefton, B. M.,Hunter, T.& Raschke, W. C. (1981) Proc. Nutl.
Acud. Sci. U.S.A. 78, 1552-1556
Wang, J. Y.J.; Queen, C. & Baltimore, D. (1982) J . Biol. Chem.
257, 13181-13184
Witte, 0.N., Dasgupta, A. &Baltimore,D. (1980) Nuture (London)
283, 826-831
Investigation of the activity of yeast phosphoglycerate kinase by site-specific mutagenesis
CARON A. B. WILSON,* MICHAEL F. TUITE,t
MELANIE J. DOBSON,t SUSAN M. KINGSMAN,?
ALAN J. KINGSMAN,? L. ANNE GLOVER,*
NORMAN HARDMAN,* HERMAN C. WATSON$
and LINDA A. FOTHERGILL*
*Department of Biochemistry, University of Aberdeen,
Marischal College, Aberdeen AB9 IAS, U.K.,
?Department of Biochemistry, University of Oxford, South
Parks Road, Oxford OX1 3QU. U . K . ,and $Department of
Biochemistry, University of Bristol, Bristol BS8 1TD, U.K.
Yeast phosphoglycerate kinase (EC 2.7.2.3) is ideally suited
for site-specific mutagenesis studies. The enzyme is particularly well characterized, both kinetically and structurally. It
is active in all cells during glycolysis, and catalyses the
formation of ATP by the transfer of a phosphoryl group
from 1,3-bisphosphoglycerate to ADP. There is a requirement for bivalent cations (Mnz+or MgZ+),since the metalion complexes are the true substrates. The high-resolution
crystallographic structure of phosphoglycerate kinase is
known (Watson et al., 1982), and the amino acid sequence
of the protein and nucleotide sequence of the gene have
been determined (Perkins et al., 1983).
Although so much is known about phosphoglycerate
kinase, certain intriguing questions remain. For example, it
is clear that the catalytic mechanism involves substantial
movement of the two domains. (The enzyme is in an 'open'
conformation for ligand binding and release, and in a
'closed' conformation for phosphoryl transfer.) How is this
triggered? Which residues are crucial for this process?
Examination of the structure of the enzyme reveals an interesting interaction between Glu-190 and His-388, which
\
P
0
Fig. 1. Properties of plasmid pMA27
(a) Partial restriction map. m,HindIII fragment containing the phosphoglycerate kinase gene (PGK); I
double
,
EcoRI fragment derived from yeast 2p
plasmid. The remainder of plasmid pMA27 consists of plasmid pBR322. (b) Tracks
1-4, agarose-gel electrophoresis of plasmid pMA27 and its restriction fragments: I ,
HindIII digest of bacteriophage 1;2, plasmid pMA27; 3, BamHI digest of plasmid
pMA27; 4, HindIII digest of plasmid pMA27. Track 5 , expression of phosphoglycerate kinase from plasmid pMA27 in yeast MD40-4C as shown by sodium
dodecyl sulphate-polyacrylamide-gel electrophoresis of cell extract. Abbreviations:
PGK, phosphoglycerate kinase; kbp, kilo-base-pairs.
1984