CHARACTERIZATION OF MEMBRANE-BOUND SERINE-PROTEASE RELATED TO DEGRADATION OF OXIDATIVELY DAMAGED ERYTHROCYTE-MEMBRANE PROTEINS

It has been shown that erythrocyte membrane proteins become susceptibl e to degradation by membrane-bound serine protease activity after oxid ative modification of the membranes (M. Beppu, M. Inoue, T. Ishikawa, K. Kikugawa, Biochim. Biophys. Acta 1196 (1994) 81-87). The aim of the present study was to clarify the presence of the serine protease in o xidized erythrocyte membranes and to characterize the selectivity of t he enzyme to oxidized proteins. Human erythrocytes were oxidized in vi tro with xanthine/xanthine oxidase/Fe(III) and oxidized membranes isol ated. Proteolytic activity of the membranes toward spectrin obtained f rom oxidized membranes and bovine serum albumin oxidized with H2O2/hor seradish peroxidase was increased by membrane oxidation, and the degra dability of the substrates was increased by substrate oxidation. The p roteolytic activity was inhibited by the serine protease inhibitor dii sopropyl fluorophosphate (DFP). The 72 kDa and 80 kDa proteins in the membranes were labeled by [H-3]DFP when detected by sodium dodecyl sul fate-polyacrylamide gel electrophoresis under reducing conditions and subsequent fluorography. The 72 kDa protein was found to be a serine e nzyme, acetylcholine esterase. The 80 kDa protein appeared to be respo nsible for the degradation of oxidatively damaged proteins. The 80 kDa protein was loosely bound to membranes and readily solubilized into a 0.1% NP-40 detergent solution. The presence of the same 80 kDa protea se in intact erythrocyte cytosol was suggested. The increased serine p rotease activity in oxidized membranes can result from the increased a dherence of the cytosolic 80 kDa serine protease to the membranes due to oxidation. 0005-2736/98/$ - see front matter (C) 1998 Elsevier Scie nce B.V. All rights reserved.