Purification and characterization of a serine protease in erythrocyte cytosol that is adherent to oxidized membranes and preferentially degrades proteins modified by oxidation and glycation

A serine protease that preferentially degrades oxidized and glycated protei ns was shown to be present in erythrocyte cytosol. Human erythrocyte cytoso l was labeled with [H-3] diiso- propyl fluorophosphate (DFP) and passed thr ough a column of carboxymethyl-Sephadex to obtain radioactive fractions fre e of hemoglobin. The fractions contained a single radioactive 80-kDa protei n, as analyzed by sodium dodecyl sulfate-polyacpylamide gel electrophoresis (PAGE)/fluorography. The radioactive 80-kDa protein bound to unoxidized er ythrocyte membranes, and more effectively to oxidized membranes. The radioa ctive protein was purified through a column of diethylaminoethyl-cellulose and by preparative native-PAGE in a purity of 80%, Antibody against the cyt osolic 80-kDa protein bound to 80-kDa protein of erythrocyte membranes, ind icating the presence of the same protein in the membrane. The antibody boun d more effectively to oxidized membranes than to unoxidized membranes, The 80-kDa protein partially purified from unlabeled cytosol degraded more effe ctively oxidized bovine serum albumin (BSA), oxidized Igf;, and glycated BS A more effectively than the corresponding unoxidized or unglycated proteins , Degradation of oxidized or glycated proteins was effectively inhibited by DFP, Hence, the protein is an 80-kDa serine protease that is adherent to o xidized membranes and is responsible for degradation of proteins modified b y oxidation and glycation.