Characterization of membrane-bound serine protease related to degradation of oxidatively damaged erythrocyte membrane proteins.
It has been shown that erythrocyte membrane proteins become susceptible to degradation by membrane-bound serine protease activity after oxidative 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 oxidized erythrocyte membranes and to characterize the selectivity of the enzyme to oxidized proteins. Human erythrocytes were oxidized in vitro with xanthine/xanthine oxidase/Fe(III) and oxidized membranes isolated. Proteolytic activity of the membranes toward spectrin obtained from oxidized membranes and bovine serum albumin oxidized with H2O2/horseradish peroxidase was increased by membrane oxidation, and the degradability of the substrates was increased by substrate oxidation. The proteolytic activity was inhibited by the serine protease inhibitor diisopropyl fluorophosphate (DFP). The 72 kDa and 80 kDa proteins in the membranes were labeled by [3H]DFP when detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and subsequent fluorography. The 72 kDa protein was found to be a serine enzyme, acetylcholine esterase. The 80 kDa protein appeared to be responsible 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 protease in intact erythrocyte cytosol was suggested. The increased serine protease activity in oxidized membranes can result from the increased adherence of the cytosolic 80 kDa serine protease to the membranes due to oxidation.
Fujino
T
T
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
Ishikawa
T
T
Inoue
M
M
Beppu
M
M
Kikugawa
K
K
D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000494:pharmacology; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012697:Serine Endopeptidases / Q000097:blood* / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism
D008565:Membrane Proteins; D015842:Serine Proteinase Inhibitors; D013049:Spectrin; D007531:Isoflurophate; D019820:Xanthine; D012710:Serum Albumin, Bovine; D014969:Xanthine Oxidase; D012697:Serine Endopeptidases