Modification of glycophorin A during oxidation of erythrocyte membrane.
Human erythrocyte ghosts were oxidized with tert-butyl hydroperoxide and subsequently treated with tritiated borohydride to label the membrane proteins modified during the membrane oxidation. From the ghosts, oxidized-and-tritiated glycophorin A was isolated and characterized. No intermolecular cross-links were observed as analyzed by sodium dodecylsulfate gel electrophoresis. But, the number of lysine residues was significantly reduced and susceptibility to proteinases such as trypsin, chymotrypsin and pronase was lower than that of control glycophorin A. Trypsinization of the oxidized-and-tritiated glycophorin A gave insoluble and soluble trypsin fragments. After dansylation, N-terminal amino acids of the trypsin-fragments were determined. Dansyl amino acids from the insoluble trypsin fragments were not identical with those from control insoluble counterparts in the membrane-spanning region of glycophorin A molecule. Fractionation by gel filtration of dansyl-soluble trypsin fragments, and the N-terminal amino acid analysis of the fractionated peptides indicated that the peptides derived from the glycosylated region located in the outside of the membrane matrix were identical with those from control soluble counterparts. The results suggest that the glycosylated outside region of glycophorin A was modified only slightly but the hydrophobic membrane-spanning region was extensively modified during membrane oxidation, most likely by oxidized lipids.
Tokyo College of Pharmacy, Japan.
M
M
Beppu
M
M
Takanashi
K
K
Murakami
T
T
Kato
K
K
Kikugawa
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000302:isolation & purification; D004910:Erythrocyte Membrane / Q000187:drug effects*; D006021:Glycophorins / Q000302:isolation & purification*; D006801:Humans; D008054:Lipid Peroxides / Q000096:biosynthesis; D010084:Oxidation-Reduction / Q000187:drug effects; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010545:Peroxides / Q000494:pharmacology*; D012795:Sialoglycoproteins / Q000302:isolation & purification*; D014357:Trypsin; D020122:tert-Butylhydroperoxide
D000596:Amino Acids; D003619:Dansyl Compounds; D006021:Glycophorins; D008054:Lipid Peroxides; D010446:Peptide Fragments; D010545:Peroxides; D012795:Sialoglycoproteins; D020122:tert-Butylhydroperoxide; D010447:Peptide Hydrolases; D014357:Trypsin