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Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.

Publication ,  Journal Article
Aiyar, VN; Hershfield, MS
Published in: Biochem J
December 15, 1985

S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8-azidoadenosine 3', 5'-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase.

Duke Scholars

Published In

Biochem J

DOI

ISSN

0264-6021

Publication Date

December 15, 1985

Volume

232

Issue

3

Start / End Page

643 / 650

Location

England

Related Subject Headings

  • Protein Kinase Inhibitors
  • Pregnancy
  • Placenta
  • Peptide Fragments
  • Ligands
  • Hydrolases
  • Humans
  • Female
  • Cyclic AMP
  • Biochemistry & Molecular Biology
 

Citation

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Aiyar, V. N., & Hershfield, M. S. (1985). Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP. Biochem J, 232(3), 643–650. https://doi.org/10.1042/bj2320643
Aiyar, V. N., and M. S. Hershfield. “Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.Biochem J 232, no. 3 (December 15, 1985): 643–50. https://doi.org/10.1042/bj2320643.
Aiyar, V. N., and M. S. Hershfield. “Covalent labelling of ligand binding sites of human placental S-adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP.Biochem J, vol. 232, no. 3, Dec. 1985, pp. 643–50. Pubmed, doi:10.1042/bj2320643.
Journal cover image

Published In

Biochem J

DOI

ISSN

0264-6021

Publication Date

December 15, 1985

Volume

232

Issue

3

Start / End Page

643 / 650

Location

England

Related Subject Headings

  • Protein Kinase Inhibitors
  • Pregnancy
  • Placenta
  • Peptide Fragments
  • Ligands
  • Hydrolases
  • Humans
  • Female
  • Cyclic AMP
  • Biochemistry & Molecular Biology