Enzymatic preparation of 32P-labeled beta-L-2',3',-dd-5'ATP and its use as a high-affinity, conformation-specific ligand for labeling adenylyl cyclases

Nucleosides Nucleotides. 1999 Apr-May;18(4-5):839-42. doi: 10.1080/15257779908041575.

Abstract

An enzymatic method was developed for the preparation of unlabeled and [beta-32P]-labeled beta-L-2',3'-dd-5'ATP from the monophosphate with near quantitative yields. beta-L-2',3'-dd-5'ATP was a competitive and potent inhibitor of adenylyl cyclases (IC5 approximately 30 nM). Upon uv-irradiation beta-L-2',3'-dd-[beta-32P]-5'ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state, noncompetitive inhibitor.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Adenylyl Cyclase Inhibitors
  • Adenylyl Cyclases / chemistry
  • Adenylyl Cyclases / metabolism*
  • Enzyme Inhibitors / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Ligands
  • Phosphorus Radioisotopes
  • Phosphorylation
  • Protein Conformation

Substances

  • Adenylyl Cyclase Inhibitors
  • Enzyme Inhibitors
  • Ligands
  • Phosphorus Radioisotopes
  • Adenosine Triphosphate
  • Adenylyl Cyclases