Identification and localization of three photobinding sites of iodoarylazidoprazosin in hamster P-glycoprotein

Eur J Biochem. 2001 May;268(9):2629-34. doi: 10.1046/j.1432-1327.2001.02155.x.


P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoprotein-containing plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125I-labeled peptides were separated by tricine/PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248-312 [transmembrane segment (TM)4 to TM5], 758-800 (beyond TM7 to beyond TM8) and 1160-1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / chemistry*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Amino Acid Sequence
  • Animals
  • Antineoplastic Agents / pharmacology
  • Azides / metabolism*
  • Binding Sites
  • CHO Cells
  • Cricetinae
  • Drug Resistance, Multiple
  • Humans
  • In Vitro Techniques
  • Iodine Radioisotopes
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Photoaffinity Labels
  • Photochemistry
  • Prazosin / analogs & derivatives*
  • Prazosin / metabolism*


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antineoplastic Agents
  • Azides
  • Iodine Radioisotopes
  • Peptide Fragments
  • Photoaffinity Labels
  • azidoprazosin
  • Prazosin