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. 1982 Oct 1;207(1):101–108. doi: 10.1042/bj2070101

9-azidoacridine, a new photoaffinity label for nucleotide- and aromatic-binding sites in proteins.

S P Batra, B H Nicholson
PMCID: PMC1153829  PMID: 7181853

Abstract

The effect of the photolytic reagent 9-azidoacridine, optionally 3H-labelled, was studied both kinetically and structurally on nine different enzymes, namely alpha-chymotrypsin, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, alanine dehydrogenase, D-amino acid oxidase, ribonuclease A, alkaline phosphatase and alpha-amylase. Dark inhibition was observed in several cases. The concentration of the inhibitor ranged from 0.2 microM to 0.67 microM and demonstrated competitive kinetics with nucleotide cofactors when present. All concentrations of inhibitor showed increased inhibition on photolysis. Examination of the oligopeptides from hydrolysis of the covalently 3H-labelled derivative in conjunction with known amino acid sequence and tertiary structure established that the primary site of interaction in those cases for which the tertiary structure was available involved the active-site region. The above results in conjunction with those obtained with the structural analogues 9-aminoacridine and 9-amino-1,2,3,4-tetrahydroacridine established that this reagent acts as a molecular probe of aromatic- and, in particular, nucleotide-binding sites. This reagent provides a further additional method for studying the nucleotide cofactor domain.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams M. J., Ford G. C., Koekoek R., Lentz P. J., McPherson A., Jr, Rossmann M. G., Smiley I. E., Schevitz R. W., Wonacott A. J. Structure of lactate dehydrogenase at 2-8 A resolution. Nature. 1970 Sep 12;227(5263):1098–1103. doi: 10.1038/2271098a0. [DOI] [PubMed] [Google Scholar]
  2. BAGLIONI C. An improved method for the fingerprinting of human hemoglobin. Biochim Biophys Acta. 1961 Apr 1;48:392–396. doi: 10.1016/0006-3002(61)90490-5. [DOI] [PubMed] [Google Scholar]
  3. Evenberg A., Meyer H., Gaastra W., Verheij H. M., De Haas G. H. Amino acid sequence of phospholipase A2 from horse pancreas. J Biol Chem. 1977 Feb 25;252(4):1189–1196. [PubMed] [Google Scholar]
  4. FRASER D. M., KERMACK W. O. The inhibitory action of some antimalarial drugs and related compounds on the hexokinase of yeast and of Plasmodium berghei. Br J Pharmacol Chemother. 1957 Mar;12(1):16–23. doi: 10.1111/j.1476-5381.1957.tb01355.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HIRS C. H. The oxidation of ribonuclease with performic acid. J Biol Chem. 1956 Apr;219(2):611–621. [PubMed] [Google Scholar]
  6. HURWITZ J., FURTH J. J., MALAMY M., ALEXANDER M. The role of deoxyribonucleic acid in ribonucleic acid synthesis. III. The inhibition of the enzymatic synthesis of ribonucleic acid and deoxyribonucleic acid by actinomycin D and proflavin. Proc Natl Acad Sci U S A. 1962 Jul 15;48:1222–1230. doi: 10.1073/pnas.48.7.1222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Harris J. I., Perham R. N. Glyceraldehyde 3-phosphate dehydrogenase from pig muscle. Nature. 1968 Sep 7;219(5158):1025–1028. doi: 10.1038/2191025a0. [DOI] [PubMed] [Google Scholar]
  8. Hartley B. S., Brown J. R., Kauffman D. L., Smillie L. B. Evolutionary similarities between pancreatic proteolytic enzymes. Nature. 1965 Sep 11;207(5002):1157–1159. doi: 10.1038/2071157a0. [DOI] [PubMed] [Google Scholar]
  9. INGRAM V. M. Abnormal human haemoglobins. I. The comparison of normal human and sickle-cell haemoglobins by fingerprinting. Biochim Biophys Acta. 1958 Jun;28(3):539–545. doi: 10.1016/0006-3002(58)90516-x. [DOI] [PubMed] [Google Scholar]
  10. KIND P. R., KING E. J. Estimation of plasma phosphatase by determination of hydrolysed phenol with amino-antipyrine. J Clin Pathol. 1954 Nov;7(4):322–326. doi: 10.1136/jcp.7.4.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Moras D., Olsen K. W., Sabesan M. N., Buehner M., Ford G. C., Rossmann M. G. Studies of asymmetry in the three-dimensional structure of lobster D-glyceraldehyde-3-phosphate dehydrogenase. J Biol Chem. 1975 Dec 10;250(23):9137–9162. doi: 10.2210/pdb1gpd/pdb. [DOI] [PubMed] [Google Scholar]
  12. Nicholson B. H., Peacocke A. R. The inhibition of ribonucleic acid polymerase by acridines. Biochem J. 1966 Jul;100(1):50–58. doi: 10.1042/bj1000050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Rossmann M. G., Adams M. J., Buehner M., Ford G. C., Hackert M. L., Lentz P. J., Jr, McPherson A., Jr, Schevitz R. W., Smiley I. E. Structural constraints of possible mechanisms of lactate dehydrogenase as shown by high resolution studies of the apoenzyme and a variety of enzyme complexes. Cold Spring Harb Symp Quant Biol. 1972;36:179–191. doi: 10.1101/sqb.1972.036.01.025. [DOI] [PubMed] [Google Scholar]
  14. Russell J., Jeng S. J., Guillory R. J. Arylazido aminopropionyl ATP, and active site directed photoaffinity reagent for mitochondrial adenosine triphosphatase. Biochem Biophys Res Commun. 1976 Jun 21;70(4):1225–1234. doi: 10.1016/0006-291x(76)91033-0. [DOI] [PubMed] [Google Scholar]
  15. SINGH A., THORNTON E. R., WESTHEIMER F. H. The photolysis of diazoacetylchymotrypsin. J Biol Chem. 1962 Sep;237:3006–3008. [PubMed] [Google Scholar]
  16. SMYTH D. G., STEIN W. H., MOORE S. The sequence of amino acid residues in bovine pancreatic ribonuclease: revisions and confirmations. J Biol Chem. 1963 Jan;238:227–234. [PubMed] [Google Scholar]
  17. Seiler N., Wiechmann J. Zum Nachweis von Aminosäuren im 10-10-Mol-Massetab. Trennung von 1-Dimethylamino-naphthalin-5-sulfonyl-aminosäuren auf Dünnschichtchromatogrammen. Experientia. 1964 Oct 15;20(10):559–560. doi: 10.1007/BF02150289. [DOI] [PubMed] [Google Scholar]
  18. Sperling J., Havron A. Photochemical cross-linking of neighboring residues in protein-nucleic acid complexes: rnase and pyrimidine nucleotide inhibitors. Biochemistry. 1976 Apr 6;15(7):1489–1495. doi: 10.1021/bi00652a020. [DOI] [PubMed] [Google Scholar]
  19. Taylor S. S. Amino acid sequence of dogfish muscle lactate dehydrogenase. J Biol Chem. 1977 Mar 10;252(5):1799–1806. [PubMed] [Google Scholar]
  20. Taylor S. S., Oxley S. S., Allison W. S., Kaplan N. O. Aminoacid sequence of dogfish M4 lactate dehydrogenase. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1790–1793. doi: 10.1073/pnas.70.6.1790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. WALLACE R. A., KURTZ A. N., NIEMANN C. INTERACTION OF AROMATIC COMPOUNDS WITH ALPHA-CHYMOTRYPSIN. Biochemistry. 1963 Jul-Aug;2:824–836. doi: 10.1021/bi00904a035. [DOI] [PubMed] [Google Scholar]
  22. Witt I., Neufang B., Muller H. The effect of proflavin in yeast cells. Biochim Biophys Acta. 1968 Nov 12;170(1):216–218. doi: 10.1016/0304-4165(68)90180-3. [DOI] [PubMed] [Google Scholar]
  23. Woronick C. L. Alcohol dehydrogenase from human liver. Methods Enzymol. 1975;41:369–374. doi: 10.1016/s0076-6879(75)41082-5. [DOI] [PubMed] [Google Scholar]

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