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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 278 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