Modification of chloride ion transport in human erythrocyte ghost membranes by photoaffinity labeling reagents based on the structure of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB).
Using human red blood cell ghost membranes, we have evaluated 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid and 5-nitro-2-[N-3-(4-azido-2,3,5,6-tetrafluorophenyl)-propylamino]- benzoic acid (FAzNPPB) as photoaffinity labeling agents based on the structure of the widely important Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB). The tetrafluoro-substituted aryl azide was found to be a more effective photoinactivating agent than the corresponding protio compound. Using a tritiated version ([3H]FAzNPPB), we demonstrated that photoinactivation of Cl- flux was accompanied by photolabeling of the band 3 protein and membrane lipids. Both processes were diminished in the presence of NPPB and the related arylanthranilate flufenamic acid. Photolabeling resulted in the incorporation of 1.0 +/- 0.2 mol 3H/mol protein in the band 3 integral membrane domain, whereas the cytoplasmic domain was essentially unlabeled. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photolabeling was found to be the result of partial labeling of at least three different regions of the membrane domain. Based on trypsin proteolysis, reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry analysis, it is proposed that one of the sites of photolabeling is the peptide lys-phe-lys (590-592). FAzNPPB is a successful polyfluoro aryl azide photoaffinity labeling agent which may be of further use in studying the diverse effects of arylanthranilates on biological membranes.
Branchini
B R
BR
Department of Chemistry, Connecticut College, New London 06320, USA.
Murtiashaw
M H
MH
Eckman
E A
EA
Egan
L A
LA
Alfano
C V
CV
Stroh
J G
JG
D000345:Affinity Labels / Q000494:pharmacology*; D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism; D001386:Azides / Q000494:pharmacology*; D001665:Binding Sites; D018118:Chloride Channels / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000187:drug effects* / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D066298:In Vitro Techniques; D017136:Ion Transport / Q000187:drug effects / Q000528:radiation effects; D008969:Molecular Sequence Data; D009579:Nitrobenzoates / Q000494:pharmacology*; D010782:Photolysis
C093116:5-nitro-2-(N-3-(4-azido-2,3,5,6-tetrafluorophenyl)propylamino)benzoic acid; D000345:Affinity Labels; D001457:Anion Exchange Protein 1, Erythrocyte; D001386:Azides; D018118:Chloride Channels; D002712:Chlorides; D009579:Nitrobenzoates; C068305:5-nitro-2-(N-3-(4-azidophenyl)propylamino)benzoic acid; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid