The anion transporter and a 28 kDa protein are selectively photolabeled by p-azidobenzylphlorizin under conditions that alter RBC morphology, flexibility, and volume.
Tritiated p-azidobenzylphlorizin (p-AzBPhz) was photoactivated in the presence of red blood cells under conditions previously found to alter morphology, flexibility and volume. When less than 0.25 million molecules were added per cell, only a 28 kDa peptide was photolabeled: at 1-2 million molecules added, band 3 also incorporated significant radioactivity. When using leaky ghosts, other proteins became labeled, including those limited to the cytoplasm. Protein N-deglycosylation caused a shift of radiolabeled band 3 to higher Rf values on SDS-PAGE gels but not for the 28 kDa band; the latter was, however, susceptible to enzymatic digestion by NANase (N-acetylneuraminidase) III but not by NANase II. Inhibition of photoincorporation into both receptors by unlabeled p-AzBPhz was dose-dependent. Mercuric chloride and p-CMBS selectively blocked 28 kDa peptide labeling. DIDS partially blocked at band 3; after 15% inhibition, greater DIDS concentrations caused increased incorporation into the 28 kDa peptide. These results, and a temperature-dependent labeling pattern, suggest that: (i) cellular changes occur when p-AzBPhz binds to the exofacial sides of the anion transporter and 28 kDa peptide; (ii) these proteins may be physically associated in the native membrane; (iii) they mediate ligand-induced changes in morphology, flexibility, and volume.
Hoefner
D M
DM
Graduate Center for Toxicology, University of Kentucky, Lexington, 40536, USA. hoefner.daniel@mayo.edu
Blank
M E
ME
Diedrich
D F
DF
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D027321:Anion Transport Proteins; D001386:Azides / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism; D010695:Phlorhizin / Q000031:analogs & derivatives* / Q000378:metabolism; D012212:Rheology
D000345:Affinity Labels; D001457:Anion Exchange Protein 1, Erythrocyte; D027321:Anion Transport Proteins; D001386:Azides; D001798:Blood Proteins; D002352:Carrier Proteins; D008024:Ligands; D008565:Membrane Proteins; C068043:erythrocyte transmembrane protein 28kDa, human; C056185:phlorizinyl 5'-benzylazide; D010695:Phlorhizin; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid