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 proteins was shown to be present in erythrocyte cytosol. Human erythrocyte cytosol was labeled with [3H]diisopropyl fluorophosphate (DFP) and passed through a column of carboxymethyl-Sephadex to obtain radioactive fractions free of hemoglobin. The fractions contained a single radioactive 80-kDa protein, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE)/fluorography. The radioactive 80-kDa protein bound to unoxidized erythrocyte membranes, and more effectively to oxidized membranes. The radioactive protein was purified through a column of diethylaminoethyl-cellulose and by preparative native-PAGE in a purity of 80%. Antibody against the cytosolic 80-kDa protein bound to 80-kDa protein of erythrocyte membranes, indicating the presence of the same protein in the membrane. The antibody bound more effectively to oxidized membranes than to unoxidized membranes. The 80-kDa protein partially purified from unlabeled cytosol degraded more effectively oxidized bovine serum albumin (BSA), oxidized IgG, and glycated BSA 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 oxidized membranes and is responsible for degradation of proteins modified by oxidation and glycation.
Fujino
T
T
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392, Japan.
Tada
T
T
Beppu
M
M
Kikugawa
K
K
D000906:Antibodies / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D007531:Isoflurophate / Q000737:chemistry; D010084:Oxidation-Reduction; D011506:Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014316:Tritium; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D011506:Proteins; D014316:Tritium; D007531:Isoflurophate; D012710:Serum Albumin, Bovine; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin