Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling

Biochim Biophys Acta. 2001 Jun 6;1512(2):215-24. doi: 10.1016/s0005-2736(01)00321-2.


To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [(3)H]diazofluorene ([(3)H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa alpha-subunit of the (Na(+)+K(+))-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by (3)H release.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Annexins / chemistry
  • Binding Sites
  • Cattle
  • Cell Membrane / chemistry*
  • Diazonium Compounds
  • Electrophoresis, Polyacrylamide Gel
  • Fluorenes
  • Fluorescent Dyes
  • Humans
  • Membrane Proteins / analysis*
  • Membrane Proteins / chemistry*
  • Molecular Sequence Data
  • Molecular Weight
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Photoaffinity Labels
  • Porins / chemistry
  • Protein Subunits
  • Pyrenes
  • Rats
  • Receptors, Nicotinic / analysis*
  • Receptors, Nicotinic / chemistry
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Sodium-Potassium-Exchanging ATPase / chemistry
  • Sodium-Potassium-Exchanging ATPase / isolation & purification
  • Torpedo
  • Tritium
  • Voltage-Dependent Anion Channels


  • Annexins
  • Diazonium Compounds
  • Fluorenes
  • Fluorescent Dyes
  • Membrane Proteins
  • Peptide Fragments
  • Photoaffinity Labels
  • Porins
  • Protein Subunits
  • Pyrenes
  • Receptors, Nicotinic
  • Voltage-Dependent Anion Channels
  • Tritium
  • azidopyrene
  • 2-diazofluorene
  • Sodium-Potassium-Exchanging ATPase