Macroxyproteinase (M.O.P.): a 670 kDa proteinase complex that degrades oxidatively denatured proteins in red blood cells.
Erythrocytes and reticulocytes are shown to undergo rapid rates of protein degradation following exposure to oxidative stress. Experiments with ATP depletion revealed that, unlike the proteolysis of many other abnormal proteins, the degradation of oxidatively modified proteins is an ATP-independent process. Ion exchange chromatography (DEAE Sepharose CL-6B), ammonium sulfate precipitation, gel filtration chromatography (Sephacryl S-300 or Sepharose CL-6B), and a second ion exchange step were used to resolve the activity responsible for degrading oxidatively modified proteins from (dialyzed) cell-free extracts of erythrocytes and reticulocytes. Gel filtration studies revealed that some 70-80% of the activity in erythrocytes, and some 60-70% of the activity in reticulocytes, is expressed by a 670 kDa proteinase complex that is not stimulated by ATP (in fact, ATP is slightly inhibitory). This proteinase complex is inhibited by sulfhydryl reagents, serine reagents, and transition metal chelators, and has a pH optimum of 7.8. We propose the trivial name "macroxyproteinase" or "M.O.P." (abbreviated from Macro-Oxy-Proteinase) for the complex because of its large size, substrate preference (oxidatively modified proteins), and inhibitor profile (which indicates multiple catalytic sites). Electrophoresis studies of the 670 kDa M.O.P. complex revealed the presence of 8 distinct polypeptide subunits with the following apparent molecular sizes: 21.5, 25.3, 26.2, 28.1, 30.0, 31.9, 33.3, and 35.7 kDa. The large molecular size of the M.O.P. complex, its ATP- and ubiquitin-independence, its inhibitor profile, its distinctive subunit banding pattern in denaturing electrophoresis gels, its pH optimum, and its proteolytic profile with fluorogenic peptide substrates all indicate that M.O.P. is identical to 600-700 kDa neutral/alkaline proteinase complexes that have been isolated from a wide variety of eucaryotic cells and tissues, but for which no function has previously been clear. We propose that macroxyproteinase is responsible for catalyzing most of the selective degradation of oxidatively denatured proteins in red blood cells. We further suggest that M.O.P. may perform the same function in other eucaryotic cells and tissues.
Pacifici
R E
RE
Institute for Toxicology, University of Southern California, Los Angeles 90033.
Salo
D C
DC
Davies
K J
KJ
D000328:Adult; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002417:Cattle; D003546:Cysteine Endopeptidases / Q000097:blood* / Q000302:isolation & purification; D004912:Erythrocytes / Q000378:metabolism*; D005609:Free Radicals; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood; D006878:Hydroxides / Q000097:blood; D017665:Hydroxyl Radical; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000097:blood* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D046988:Proteasome Endopeptidase Complex; D011489:Protein Denaturation; D011817:Rabbits
D001798:Blood Proteins; D005609:Free Radicals; D006878:Hydroxides; D009097:Multienzyme Complexes; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex