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.
Highlights
Studies were carriedout to characterize further the nized and repaired
F.) from the Amer-The studies presented here further characterize the ATPindependent system in intact erythrocytes and extracts that degrades oxidanttreated hemoglobin (Ox-Hb).By immunoprecipitating certainsoluble proteolytic enzymesand by using a variety of general and specific inhibitors of proteases, we provide data suggesting that the ican Heart Association
Aliquots were supplemented with the chloride salts of the divalentcations listed above, all at 50 p ~ A.fter 1 h at room temperature, the hreakdown of insulin and Ox-Hb was measured as described in Fig
Summary
Chemicals-Casein, porcine insulin, N-ethylmaleimide, BSA, alanine dehydrogenase, phenylhydrazinPe,CMBS, iodoacetamide, EDTA, EGTA, o-phenanthroline, ascorbic acid, sodium azide, phenylmethanesulfonyl fluoride, phosphoramidon, and poly(G1u-Tyr) After washing the erythrocytes five times in ice-cold saline, unless otherwise indicated extracts were prepared by lysing the cells in 2 volumes of 1 mM DTT and removing the membranes by centrifugation (30,000 X g, 1 h).Supernatants were dialyzed overnight against 20 mM Tris-HCI, 20 mM KCI, 1mM MgC12, 0.5mM. Alanine was quantitated by a sensitive fluorometric assay (Williamson, 1974).The breakdown of oxidatively damaged hemoglobin was linear with time (up to 2 h) and with the amount of lysate added (data not shown) using either the release of alanine or radiolabeled polypeptide to measure proteolysis. After 16 h at 4 "C, 200 pl of protein A-Sepharose beads coated with rabbit anti-murine IgG (Calbiochem) was added to each mixture and incubated for 2 h. The membrane was incubated overnight with a 0.1 mg/ml solution of monoclonal antibody, and the antigen-antibody complex was detected with goat anti-murine IgG coupled to alkaline phosphatase using a chemiluminescent substrate (Bio-Rad). The signal was detected after a 10min exposure on x-ray film
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