Presence of membrane-bound proteinases that preferentially degrade oxidatively damaged erythrocyte membrane proteins as secondary antioxidant defense.
Human erythrocytes were oxidized with xanthine/xanthine oxidase/ferric ion or ADP/ferric ion at 37 degrees C for several hours. Band 3 protein and spectrin of the oxidized cells were found to be significantly modified as analyzed by radiolabeling with tritiated borohydride. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the xanthine/xanthine oxidase/ferric iron-oxidized cells and subsequent immunoblotting with anti band 3 protein showed that band 3 protein was fragmented into smaller molecular-weight fragments. When the cell membrane obtained from the oxidized cells were incubated at pH 7.4 and 37 degrees C for several hours in the presence of alpha-tocopherol, extensive degradation of band 3 protein and spectrin was observed. Band 3 protein was found to be most susceptible to the degradation. Degradation of band 3 protein was also observed after similar incubation of the membrane from the ADP/ferric ion-oxidized cells. Membrane-bound serine- and metalloproteinases were responsible for the degradation of band 3 protein, because the degradation was remarkably inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, and partially by ethylenediaminetetraacetic acid. Hence, the membrane proteins became susceptible to membrane-bound proteinases by oxidative stress. This observation suggests that these membrane-bound proteinases exist to remove oxidatively damaged proteins from the cell membrane.
Beppu
M
M
Tokyo College of Pharmacy, Japan.
Inoue
M
M
Ishikawa
T
T
Kikugawa
K
K
D001457:Anion Exchange Protein 1, Erythrocyte / Q000032:analysis; D000975:Antioxidants / Q000378:metabolism*; D010450:Endopeptidases / Q000032:analysis*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D018384:Oxidative Stress; D011480:Protease Inhibitors / Q000494:pharmacology*; D013049:Spectrin / Q000032:analysis
D001457:Anion Exchange Protein 1, Erythrocyte; D000975:Antioxidants; D008565:Membrane Proteins; D011480:Protease Inhibitors; D013049:Spectrin; D010450:Endopeptidases