Biotin-conjugated reagents as site-specific probes of membrane protein structure: application to the study of the human erythrocyte hexose transporter.
A novel labeling procedure using biotin-conjugated protein-modifying reagents has been employed to study the structure and function of the human erythrocyte hexose transporter. The carbohydrate moiety of the isolated, reconstituted transporter was labeled by using galactose oxidase/biotin hydrazide. Cysteine residues, which are essential for transporter function, were tagged with a biotin-conjugated maleimide. Labeling with this reagent inhibited the binding of cytochalasin B to the transporter. Following sodium dodecyl sulfate-gel electrophoresis, labeling of the transporter and its proteolytic fragments was detected by Western blotting and probing with alkaline phosphatase-conjugated avidin. After tryptic cleavage of the transporter into two membrane domains, preparations reacted with galactose oxidase/biotin hydrazide were labeled on the 25-kDa glycosylated fragment, but not on the carbohydrate-free 19-kDa peptide. Biotin-maleimide-labeled cysteine residues on both peptides. Transporter polypeptide was fragmented more extensively using Staphylococcus aureus V8 protease. Limited digestion produced a broad band of 30-50 kDa and sharper bands of 23 and 21 kDa. More extensive digestion resulted in the disappearance of the 23-kDa peptide and the appearance of sharp bands of 20, 19, 17, 13, 11, 8, and 7 kDa. Biotin label introduced with galactose oxidase/biotin hydrazide was found on the broad 30-kDa band, confirming its identity as a glycopeptide. All of the peptides weighing more than 11 kDa contained cysteine residues labeled with biotin maleimide, while the 8- and 7-kDa peptides were unlabeled. These results demonstrate the potential usefulness of biotin-conjugated reagents as site-specific probes of membrane protein structure.
Deziel
M R
MR
Department of Medicine, State University of New York, Buffalo 14215.
Mau
M M
MM
D001710:Biotin; D002352:Carrier Proteins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology; D005692:Galactose Oxidase / Q000737:chemistry*; D006601:Hexoses / Q000737:chemistry*; D006801:Humans; D006834:Hydrazines; D006868:Hydrolysis; D008301:Maleimides; D008565:Membrane Proteins / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D006601:Hexoses; D006834:Hydrazines; D008301:Maleimides; D008565:Membrane Proteins; C043592:maleimide; D001710:Biotin; D005692:Galactose Oxidase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D003545:Cysteine