Abstract
Torpedo californica acetylcholine receptor (AcChR) enriched, sealed vesicles have been specifically labeled on the cytoplasmic surface with pyridoxal 5′-phosphate (Perez-Ramirez, B., and Martinez-Carrion, M., 1989,Biochemistry 28, 5034–5040). After chromatography of the peptide fragments produced by tryptin digestion of labeled AcChR, several fractions containing the phosphopyridoxyl label were obtained. Edman degradation identified one of the fractions, with sequence SRSELMFEKQSER, as corresponding to residues 377–389 in theδ subunit (primary structure). The latter must be a cytoplasmic region of this transmembranous protein, and residueδK385 must reside in a water-soluble exposed domain of the cytosolic side of the membrane. Introduction of phosphopyridoxyl residues allows for their potential use as probes of conformational changes in the cytosolic surface of the receptor molecule.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- AcChR:
-
acetylcholine receptor
- PLP:
-
pyridoxal 5′-phosphate
- PNP:
-
pyridoxine 5′-phosphate
- α-Bgt:
-
α-bungarotoxin
- OG:
-
Β-D-octylglucopyranoside
- TPCK:
-
N-tosyl-L-phenylalanine chloromethyl ketone
- TFA:
-
trifluoroacetic acid
- HPLC:
-
high-performance liquid chromatography
- RP-HPLC:
-
reversed-phase high-performance liquid chromatography
- HEPES:
-
N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]
- PBS:
-
10 mM sodium phosphate, pH 7.5, 100 mM NaCl
- SDS-PAGE:
-
sodium dodecyl sulfate polyacrylamide gel electrophoresis
- PTH:
-
phenylthiohydantoin
References
Baezinger, J. E., Miller, K. W., and Rothchild, K. J. (1992).Biophys. J. 61, 983–992.
Baezinger, J. E., Miller, K. W., and Rothchild, K. J. (1993).Biochemistry 32, 5448–5454.
Bowers-Komro, D. M., Hagen, T. M., and McCormick, D. B. (1986).Methods Enzymol. 122, 116–119.
Chattopadhyay, A., and McNamee, M. G. (1991).Biochemistry 30, 7159–7164.
Churchich, J. E. (1965).Biochemistry 10, 1405–1410.
Claudio, T., Ballivet, M., Patrick, J., and Heinemann, S. (1983).Proc. Natl. Acad. Sci. USA 80, 1111–1115.
Di Paolo, M., Czajkowski, C., and Karlin, A. (1989).J. Biol. Chem. 264, 15,457–15,463.
Devillers-Thiery, A., Giraudat, J., Bantaboulet, M., and Changeux, J. P. (1983).Proc. Natl. Acad. Sci. USA 80, 2067–2071.
Dwyer, B. P. (1991).Biochemistry 30, 4105–4112.
Eftink, M. R., and Ghiron, C. A. (1976a).J. Phys. Chem. 80, 486–493.
Eftink, M. R., and Ghiron, C. A. (1976b).Biochemistry 15, 672–680.
Elliot, J., Dunn, S. M. J., Blanchard, S. G., and Raftery, M. A. (1979).Proc. Nat. Acad. Sci. USA 76, 2576–2580.
Farach, M. C., Mihovilovic, M., Paraschos, A., and Martinez-Carrion, M. (1982).Arch. Biochem. Biophys. 214, 140–154.
Farach, M. C., and Martinez-Carrion, M. (1983).J. Biol. Chem. 258, 4166–4170.
Finer-Moore, J., and Stroud, R. M. (1984).Proc. Natl. Acad. Sci. USA 81, 155–159.
Fong, T. M., and McNamee, M. G. (1987).Biochemistry 26, 3871–3880.
Garcia-Borron, J. C., Bieber, A., and Martinez-Carrion, M. (1987).Biochemistry 26, 4295–4303.
Giraudat, J., Montecucco, C., Bisson, R., and Changeux, J. P. (1985).Biochemistry 24, 3121–3127.
Gonzalez-Ros, J. M., Paraschos, A., and Martinez-Carrion, M. (1980).Proc. Nat. Acad. Sci. USA 77, 1796–1800.
Gonzalez-Ros, J. M., Paraschos, A., Farach, M. C., and Martinez-Carrion, M. (1981).Biochim. Biophys. Acta 643, 407–420.
Gonzalez-Ros, J. M., Farach, M. C., and Martinez-Carrion, M. (1983).Biochemistry 22, 3807–3811.
Görne-Tschelnokow, V., Hucho, F., Naumann, D., Barth, A., and MÄntele, W. (1992).FEBS Lett. 309, 213–217.
Guy, H. R. (1984).Biophys. J. 45, 249–261.
Heidmann, T., Oswald, R., and Changeux, J. P. (1983).Biochemistry 22, 3112–3127.
Lakowicz, J. (1983). InPrinciples of Fluorescence Spectroscopy, Plenum Press, New York, pp. 303–339.
Lee, T., Witzemann, V., Schimerlik, M., and Raftery, M. A. (1977).Arch. Biochem. Biophys. 83, 57–63.
Lehrer, S. S., and Leavis, P. C. (1978).Methods in Enzymology 49, 226–236.
Lei, S., Raftery, M. A., and Conti-Tronconi, B. M. (1993).Biochemistry 32, 91–100.
Lowry, O. H., Rosembrough, N. J., Farr, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193, 265–275.
Maelicke, A., Plummer-Wiek, R., Fels, G., Spencer, S. R., Engelhard, M., Veltel, D., and Conti-Tronconi, B. M. (1989).Biochemistry 28, 1396–1403.
Martinez-Carrion, M., Sator, V., and Raftery, M. A. (1975).Biochem. Biophys. Res. Comm. 65, 129–137.
McCarthy, M. P., and Stroud, R. M. (1989).Biochemistry 28, 40–48.
Middlemas, D. S., and Raftery, M. A. (1987).Biochemistry 26, 1219–1223.
Mitra, A. K., McCarthy, M. P., and Stroud, R. M. (1989).J. Cell. Biol. 109, 755–774.
Moore, W. M., and Brady, R. N. (1977).Biochim. Biophys. Acta. 498, 331–335.
Naumann, D., Schultz, C., Görne-Tschelnokow, V., and Hucho, F. (1993).Biochemistry 32, 3162–3168.
Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotami, S., Futunami, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983).Nature (London) 302, 528–532.
Pedersen, S. E., and Cohen, J. B. (1990).Proc. Natl. Acad. Sci. USA 87, 2785–2789.
Perez-Ramirez, B., and Martinez-Carrion, M. (1989).Biochemistry 28, 5034–5040.
Raftery, M. A., Hunkapiller, M. W., Strader, C. D., and Hood, L. E. (1980).Science 208, 1454–1457.
Ratnam, M., Nguyen, D. L., Rivier, J., Sargent, P. B., and Lindstrom, J. (1986).Biochemistry 25, 2633–2643.
Reynolds, J. A., and Karlin, A. (1978).Biochemistry 17, 2035–2038.
Schmidt, J., and Raftery, M. A. (1973).Anal. Biochem. 52, 349–354.
Shroeder, W., Meyer, H. E., Buchner, K., Bayer, H., and Hucho, F. (1991).Biochemistry 30, 3583–3588.
Sine, S. M., and Claudio, T. (1991).J. Biol. Chem. 266, 19,369–19,377.
Soler, G., Farach, C. M., Farach, H. A., Mattingly, J. R., and Martinez-Carrion, M. (1983).Arch. Biochem. Biophys. 225, 872–878.
Tao, T., and Cho, J. (1979).Biochemistry 18, 2759–2765.
Unwind, N. (1993).Cell 72, 31–41.
Wagner, K., Edson, K., Heginbotham, L., Post, M., Huganir, R. L., and Czernik, A. J. (1991).J. Biol. Chem. 266, 23,784–23,789.
Weber, M., David-Pfeuty, T., and Changeux, J. P. (1975).Proc. Nat. Acad. Sci. USA 72, 3443–3447.
White, B. J., and Cohen, J. B. (1988).Biochemistry 27, 8741–8751.
White, B. J., and Cohen, J. B. (1992).J. Biol. Chem. 267, 15,770–15,783.
Yee, G. H., and Huganir, R. L. (1987).J. Biol. Chem. 262, 16,748–16,753.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Perez-Ramirez, B., Iriarte, A. & Martinez-Carrion, M. Residues 377-389 from the δ subunit ofTorpedo californica acetylcholine receptor are located in the cytoplasmic surface. J Protein Chem 13, 67–76 (1994). https://doi.org/10.1007/BF01891994
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01891994