Abstract
The technique of affinity labeling allows the direct identification of the sites of interaction between two molecules, for example, between substrate and enzyme or, just as well, between a drug and its target. In general, a chemically reactive group is introduced into a molecule, which then can react preferentially and irreversibly with a properly oriented amino acid functional group in the binding region of the protein(s). We applied this technique to the antibiotics chloramphenicol, puromycin and streptomycin in order to find out, which ribosomal protein is associated with the binding sites of these antibiotics. All three antibiotics were modified with bromoor iodoacetic acid such that they did not lose their known antibiotic properties. However, the introduction of a haloacetyl group into the antibiotics leads to a chemical linkage between the antibiotic and its ribosomal binding site. The respective labeled ribosomal proteins were isolated and, thus, the ribosomal protein(s) involved in the binding of the antibiotic, could be identified.
Paper No. 10 on “Affinity Labeling of Ribosomes”. Preceding paper: Pongs, O., & Lanka, E. (1974) Proc. Nat. Acad. Sci. USA, submitted for publication.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Bald, R., V.A. Erdmann and O. Pongs: Irreversible binding of chloramphenicol analogues to E.coli ribosomes. FEBS Lett. 28, 149–152 (1972).
Erdmann, V.A., S. Fahnestock, K. Higo and M. Nomura: Role of 5S RNA in the functions of 50S ribosomal subunits. Proc. Nat. Acad. Sci. USA 68, 2932–2936 (1971).
Hershey, J.W.B., E. Remold-O’Donnell, D. Kolakofsky, K.F. Dewey and R.E. Thach: Isolation and Purification of Initiation factors. Methods in Enzymology 20, 235–247 (1971).
Kaltschmidt, E. and H.G. Wittmann: Ribosomal proteins. XII.: Number of proteins in small and large ribosomal subunits of Escherichia coli as determined by two-dimensional gel electrophoresis. Proc. Nat. Acad. Sci. USA 67, 1276–1282 (1970).
Lelong, J.C., M.A. Cousin, D. Gros, M. Grunberg-Manago and F. Gros: Streptomycin induced release of fmet-tRNA from the ribosomal initiation complex. Biochem. Biophys. Res. Commun. 42, 530–537 (1971).
Modolell, J. and B.D. Davies: Breakdown by Streptomycin of Initiation Complexes Formed on Ribosomes of Escherichia coli. Proc. Nat. Acad. Sci. USA 67, 1148–1155 (1970).
Moore, P.B.: Reaction of N-ethyl maleimide with the Ribosomes of Escherichia coli. J. Mol. Biol. 60, 169–184 (1971).
Morrison, C.A., R.A. Garrett, H. Zeichhardt and G. Stöffler: Proteins occurring at, or near, the subunit interface of E.coli ribosomes. Molec. Gen. Genet., in press (1974).
Nierhaus, D. and K. Nierhaus: Identification of the chloramphenicolbinding protein in Escherichia coli ribosomes by partial reconstitution. Proc. Nat. Acad. Sci. USA 70, 2224–2228 (1973).
Nomura, M. and J.D. Watson: Ribonucleoprotein particles within chloromycetin inhibited E.coZi. J. Mol. Biol. 1, 204–217 (1959).
Pestka, S.: Inhibitors of Ribosome Functions. Ann. Rev. Microbiol. 25, 487–562 (1971).
Pongs, O., R. Bald and V.A. Erdmann: Identification of Chloramphenicol- Binding Protein in Escherichia coli Ribosomes by Affinity Labeling. Proc. Nat. Acad. Sci. USA 70, 2229–2233 (1973a).
Pongs, O., R. Bald, T. Wagner and V.A. Erdmann: Irreversible Binding of N-iodoacetylpuromycin to E.coli Ribosomes. FEBS Lett. 35, 137–140 (1973b).
Pongs, O. and V.A. Erdmann: Affinity Labeling of E.coli ribosomes with a Streptomycin Analog. FEBS Lett. 35, 137–140 (1973).
Pongs, O., K.H. Nierhaus, V.A. Erdmann and H.G. Wittmann: Active Sites in Escherichia coli Ribosomes. FEBS Lett. 40, 528–537 (1974).
Randall-Hazelbauer, L.L. and C.G. Kurland: Identification of three 30S proteins contributing to the ribosomal A-site. Molec. Gen. Genet. 115, 234–242 (1972).
Reinwald, E. and O. Pongs: Identification of ribosomal proteins associated with the streptomycin binding site by affinity labeling. Manuscript in preparation (1974).
Stahl, J., K. Dressler and H. Bielka: Studies on proteins of animal ribosomes. XVIII. Affinity labeling of rat liver ribosomes by Nbromoacetylpuromycin. FEBS Lett., in press (1974).
Vazquez, D.: The binding of chloramphenicol by ribosomes from Bacillus megaterium. Biochem. Biophys. Res. Commun. 15, 464–468 (1964).
Welfle, H., J. Stahl and H. Bielka: Studies on Proteins of Animal Ribosomes. Biochim. Biophys. Acta 243, 416–428 (1971).
Wolfe, D. and F.E. Hahn: Effects of chloramphenicol upon a ribosomal amino acid polymerization system and its binding to bacterial ribosomes. Biochim. Biophys. Acta 95, 146–155 (1965).
Yarmolinski, M.B. and G. de laHaba: Inhibition by Puromycin of Amino Acid Incorporation into Protein. Proc. Nat. Acad. Sci. USA 45, 1721–1729 (1959).
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1975 Springer-Verlag/Wien
About this paper
Cite this paper
Pongs, O., Bald, R., Erdmann, V.A., Reinwald, E. (1975). Studies on Active Sites of Ribosomes with Haloacetylated Antibiotic Analogs. In: Drews, J., Hahn, E. (eds) Drug Receptor Interactions in Antimicrobial Chemotherapy. Topics in Infectious Diseases, vol 1. Springer, Vienna. https://doi.org/10.1007/978-3-7091-8405-9_13
Download citation
DOI: https://doi.org/10.1007/978-3-7091-8405-9_13
Publisher Name: Springer, Vienna
Print ISBN: 978-3-7091-8407-3
Online ISBN: 978-3-7091-8405-9
eBook Packages: Springer Book Archive