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. 1978 Mar;75(3):1199–1203. doi: 10.1073/pnas.75.3.1199

Fluorescent photoaffinity labeling: Adenosine 3′,5′-cyclic monophosphate receptor sites

Gideon Dreyfuss *, Kenneth Schwartz *, Elkan R Blout *,†, Jorge R Barrio ‡, Fu-Tong Liu ‡, Nelson J Leonard ‡
PMCID: PMC411437  PMID: 206888

Abstract

An approach to the study of protein receptor sites in protein mixtures or supramolecular assemblies by using fluorescence spectroscopy is described. This approach, fluorescent photoaffinity labeling, combines the merits of photoaffinity labeling to attain site-directed reactivity with the probing power of fluorescent ligands. A fluorescent photoaffinity label for cyclic AMP receptor sites of cyclic AMP-dependent protein kinases was synthesized in both unlabeled and radioactive forms. The probe, 8-azido-1,N6-ethenoadenosine 3′,5′-cyclic monophosphate, mimics cyclic AMP in its ability to stimulate the phosphotransferase activity of the protein kinases and strongly competes with cyclic AMP for its binding sites in all preparations so far tested. Photolysis, after equilibration of protein kinase and 8-azido-1,N6-ethenoadenosine 3′,5′-cyclic monophosphate in the dark, effects binding of the intermediate nitrene irreversibly and specifically to the cyclic AMP sites with the development of fluorescence. Excess reagent and low molecular weight photolytic products are removable by dialysis. Studies of a crude beef heart preparation containing cyclic AMP-dependent protein kinase suggest that the cyclic AMP binding sites are hydrophobic in nature and strongly immobilize the adenine moiety of the cyclic nucleotide.

Keywords: specific binding, fluorescent probes, ethenoadenosine, photoattachment, protein kinases

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Selected References

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  1. Barrio J. R., Sattsangi P. D., Gruber B. A., Dammann L. G., Leonard N. J. Species responsible for the fluorescence of 3,N4-ethenocytidine. J Am Chem Soc. 1976 Nov 10;98(23):7408–7414. doi: 10.1021/ja00439a049. [DOI] [PubMed] [Google Scholar]
  2. Brunswick D. J., Cooperman B. S. Photo-affinity labels for adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1801–1804. doi: 10.1073/pnas.68.8.1801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gilman A. G. A protein binding assay for adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1970 Sep;67(1):305–312. doi: 10.1073/pnas.67.1.305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Guthrow C. E., Rasmussen H., Brunswick D. J., Cooperman B. S. Specific photoaffinity labeling of the adenosine 3':5'-cyclic monophosphate receptor in intact ghosts from human erythrocytes. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3344–3346. doi: 10.1073/pnas.70.12.3344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Haley B. E., Hoffman J. F. Interactions of a photo-affinity ATP analog with cation-stimulated adenosine triphosphatases of human red cell membranes. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3367–3371. doi: 10.1073/pnas.71.9.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Haley B. E. Photoaffinity labeling of adenosine 3',5'-cyclic monophosphate binding sites of human red cell membranes. Biochemistry. 1975 Aug 26;14(17):3852–3857. doi: 10.1021/bi00688a018. [DOI] [PubMed] [Google Scholar]
  7. Ikehara M., Uesugi S. Studies on nucleosides and nucleotides. 38. Synthesis of 8-bromoadenosine nucleotides. Chem Pharm Bull (Tokyo) 1969 Feb;17(2):348–354. doi: 10.1248/cpb.17.348. [DOI] [PubMed] [Google Scholar]
  8. Keeler E. K., Campbell P. A fluorescent photo-affinity label for cyclic AMP binding proteins. Biochem Biophys Res Commun. 1976 Sep 20;72(2):575–580. doi: 10.1016/s0006-291x(76)80079-4. [DOI] [PubMed] [Google Scholar]
  9. Koberstein R. 8-Azidoacenine analogs of NAD+ and FAD. Synthesis and coenzyme properties with NAD+-dependent and FAD-dependent enzymes. Eur J Biochem. 1976 Aug 1;67(1):223–229. doi: 10.1111/j.1432-1033.1976.tb10653.x. [DOI] [PubMed] [Google Scholar]
  10. Koberstein R., Cobianchi L., Sund H. Interaction of the photoaffinity label 8-azido-ADP with glutamate dehydrogenase. FEBS Lett. 1976 Apr 15;64(1):176–180. doi: 10.1016/0014-5793(76)80277-3. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Leonard N. J., Tolman G. L. Fluorescent nucleosides and nucleotides. Ann N Y Acad Sci. 1975 Aug 8;255:43–58. doi: 10.1111/j.1749-6632.1975.tb29212.x. [DOI] [PubMed] [Google Scholar]
  14. Pomerantz A. H., Rudolph S. A., Haley B. E., Greengard P. Photoaffinity labeling of a protein kinase from bovine brain with 8-azidoadenosine 3',5'-monophosphate. Biochemistry. 1975 Aug 26;14(17):3858–3862. doi: 10.1021/bi00688a019. [DOI] [PubMed] [Google Scholar]
  15. Rubin C. S., Rosen O. M. Protein phosphorylation. Annu Rev Biochem. 1975;44:831–887. doi: 10.1146/annurev.bi.44.070175.004151. [DOI] [PubMed] [Google Scholar]
  16. Secrist J. A., 3rd, Barrio J. R., Leonard N. J., Villar-Palasi C., Gilman A. G. Fluorescent modification of adenosine 3',5'-monophosphate: spectroscopic properties and activity in enzyme systems. Science. 1972 Jul 21;177(4045):279–280. doi: 10.1126/science.177.4045.279. [DOI] [PubMed] [Google Scholar]
  17. Secrist J. A., 3rd, Barrio J. R., Leonard N. J., Weber G. Fluorescent modification of adenosine-containing coenzymes. Biological activities and spectroscopic properties. Biochemistry. 1972 Sep 12;11(19):3499–3506. doi: 10.1021/bi00769a001. [DOI] [PubMed] [Google Scholar]
  18. Spencer R. D., Weber G., Tolman G. L., Barrio J. R., Leonard N. J. Species responsible for the fluorescence of 1:N6-ethenoadenosine. Eur J Biochem. 1974 Jun 15;45(2):425–429. doi: 10.1111/j.1432-1033.1974.tb03566.x. [DOI] [PubMed] [Google Scholar]
  19. Stryer L. Fluorescence spectroscopy of proteins. Science. 1968 Nov 1;162(3853):526–533. doi: 10.1126/science.162.3853.526. [DOI] [PubMed] [Google Scholar]

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