title: The ATP-independent pathway in red blood cells that degrades oxidant-damaged hemoglobin.
abstract: Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.
authors.affiliation: Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey 08903.
authors.forename: J M
authors.initials: JM
authors.lastname: Fagan
authors.forename: L
authors.initials: L
authors.lastname: Waxman
mesh_terms: D000255:Adenosine Triphosphate / Q000097:blood*; D000818:Animals; D001386:Azides / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002413:Cations, Divalent; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D006454:Hemoglobins / Q000187:drug effects* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D016877:Oxidants / Q000494:pharmacology*; D010659:Phenylhydrazines / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D011817:Rabbits; D051381:Rats
chemical_list: D001386:Azides; D002413:Cations, Divalent; D015853:Cysteine Proteinase Inhibitors; D006454:Hemoglobins; D016877:Oxidants; D010659:Phenylhydrazines; D011744:Pyrimidinones; C030299:phenylhydrazine; C026511:divicine; D000001:Calcimycin; D015759:Ionomycin; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D007930:Leucine