Inhibition of Bax channel-forming activity by Bcl-2

Science

Volumn 277, Issue 5324, 1997, Pages 370-372

  Antonsson, Bruno ^a   Conti, Franco ^b   Ciavatta, Annamaria ^b   Montessuit, Sylvie ^a   Lewis, Shareta ^a   Martinou, Isabelle ^a   Bernasconi, Lilia ^a   Bernard, Alain ^a   Mermod, Jean Jacques ^a   Mazzei, Gonzalo ^a   Maundrell, Kinsey ^a   Gambale, Franco ^b   Sadoul, Rémy ^a   Martinou, Jean Claude ^a  

^a GLAXOSMITHKLINE   (United States)

^b UNIVERSITY OF GENOA   (Italy)

Author keywords

[No Author keywords available]

Indexed keywords

CARBOXYFLUORESCEIN; CELL MEMBRANE PROTEIN; PROTEIN BAX; PROTEIN BCL 2;

ANIMAL CELL; ARTICLE; ERYTHROCYTE; LIPID MEMBRANE; NONHUMAN; PH; PRIORITY JOURNAL; PROTEIN FAMILY; PROTEIN LOCALIZATION; PROTEIN PROTEIN INTERACTION; SHEEP;

ANIMALS; APOPTOSIS; BCL-2-ASSOCIATED X PROTEIN; CELL MEMBRANE PERMEABILITY; CELLS, CULTURED; ERYTHROCYTES; FLUORESCEINS; HEMOLYSIS; HUMANS; HYDROGEN-ION CONCENTRATION; ION CHANNELS; LIPID BILAYERS; LIPOSOMES; MEMBRANE POTENTIALS; NEURONS; PATCH-CLAMP TECHNIQUES; PROTO-ONCOGENE PROTEINS; PROTO-ONCOGENE PROTEINS C-BCL-2; SHEEP; SYMPATHETIC NERVOUS SYSTEM;

EID: 9844257587     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5324.370     Document Type: Article

References (19)

1. S. J. Korsmeyer, Trends Genet. 11, 101 (1995)
2. S. W. Muchmore et al., Nature 381, 335 (1996).
3. A. J. Minn et al., ibid. 385, 353 (1997).
4. S. L. Schendel et al., Proc. Natl. Acad. Sci. U.S.A. 94, 5113 (1997).
5. S. Haldar et al., Arch. Biochem. Biophys. 315, 483 (1994).
6. C. Borner et al., J. Cell. Biol. 126, 1059 (1994).
7. Z. N. Oltvai, C. L. Milliman, S. J. Korsmeyer, Cell 74, 609 (1993).
8. Bcl-2 can interact with Bax in solution (B. Antonsson, unpublished data).
9. F. Conti, A. M. Ciavatta, F. Gambale, unpublished data.
10. T. Hennet, G. Bertoni, C. Richter, E. Peterhans, Cancer Res. 53, 1456 (1993).
11. P. Marchetti et al., J. Exp. Med. 184, 1155 (1996).
12. T. L Deckwerth et al., Neuron 17, 401 (1996).
13. I. Garcia, I. Martinou, Y. Tsujimoto, J.-C. Martinou, Science 258, 302 (1992).
14. I. Martinou et al., J. Cell Biol. 128, 201 (1995).
15. Human Bax-α lacking 20 amino acids at the COOH-terminus was expressed as a GST fusion protein or a His-tagged protein in Escherichia coli, and the protein was purified from the soluble cell fraction (B. Antonsson, in preparation). In brief, the GST-Bax fusion protein was applied to a glutathione-Sepharose column, and Bax was released by cleavage with thrombin (0.6 U/ml). Bax was subsequently purified on heparin-Sepharose, followed by fast protein liquid chromatography (FPLC) Mono Q. His-tagged Bax was purified on a Ni-nitrilotriacetic acid-agarose column followed by FPLC Mono Q. Human Bcl-2 lacking 34 amino acids at the COOH-terminus was expressed in E. coli. Bcl-2 was purified from the soluble cell fraction by sequential chromatography on Q-Sepharose, phenyl-Sepharose, heparin-Sepharose, and FPLC Mono Q. The COOH-terminal truncations were necessary because yields and solubility of full-length recombinant protein were too low.
16. 2 [B. Kenny, C. Cherveaux, I. A. Holland, Mol. Microbiol. 11, 99 (1994)].
17. Liposomes containing 20 mM 6,7-carboxyfluorescein (Sigma) were prepared as described [R. Sadoul, M. Hirn, H. Deagostini-Bazin, G. Rougon, C. Goridis, Nature 304, 347 (1983)] with 400 μg of phosphati-dylserine from bovine brain (Sigma), 400 μg of phosphatidylcholine (Sigma), and 230 μg of cholesterol (Fluka). The liposomes were dialyzed for 24 hours against phosphate-buffered saline (PBS) and diluted to 8 ml. For analysis at acidic pH, fluorescence of released dye was measured after adjustment of pH to 7.5 by addition of 1 M tris-HCI (pH 7.5).
18. 4-NaOH, 10 mM sodium citrate, 125 mM NaCl, 0.5 mM EDTA (pH 7.0) and 10 mM sodium citrate, 125 mM NaCl (pH 4.0). Membrane currents were recorded under voltage-control by a patch-clamp amplifier (EPC-7, List, Darmstadt, Germany). Voltage stimulation and data acquisition were controlled by a Macintosh microcomputer (Cupertino, CA) interfaced to the recording amplifier with a 16-bit AD/DA converter (Instrutech, Elmond, NY). Off-line analysis was done with special purpose IGOR software (Wavemetrics).
19. We thank S. Arkinstall, J. Delamarter, M. Edgerton, J. Knowles, A.-M. Surprenant, G. Turcatti, and T. Wells for critical review and helpful comments; R. Brown for Bax cDNA; K. Rose for mass spectroscopy; O. Moran for advice and help in development of analysis programs; E. Magnenat and M. Missotten for technical support; and C. Hebert for art work.