Enzymatic removal of oxidized protein aggregates from erythrocyte membranes.
Erythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1 mM Fe(III) at 37 degrees C for 3 h, the aggregation of nonionic detergent C(12)E(8)-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37 degrees C for 18 h caused reduction of the membrane protein aggregates and the (125)I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.
Fujino
T
T
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Ando
K
K
Beppu
M
M
Kikugawa
K
K
D001457:Anion Exchange Protein 1, Erythrocyte / Q000276:immunology / Q000378:metabolism; D000906:Antibodies; D015153:Blotting, Western; D003902:Detergents; D004905:Erythrocyte Aging; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology*; D008565:Membrane Proteins / Q000097:blood / Q000378:metabolism; D010084:Oxidation-Reduction; D011480:Protease Inhibitors / Q000494:pharmacology*; D012697:Serine Endopeptidases / Q000097:blood* / Q000187:drug effects*; D013997:Time Factors
D001457:Anion Exchange Protein 1, Erythrocyte; D000906:Antibodies; D003902:Detergents; D008565:Membrane Proteins; D011480:Protease Inhibitors; D007531:Isoflurophate; D012697:Serine Endopeptidases