title: Purification of a protease in red blood cells that degrades oxidatively damaged haemoglobin.
abstract: Haemoglobin damaged by exposure of red blood cells to oxidants is rapidly degraded by a proteolytic pathway which does not require ATP [Fagan, Waxman & Goldberg (1986) J. Biol. Chem. 261, 5705-5713]. By fractionating erythrocyte lysates, we have purified two proteases which hydrolyse oxidatively damaged haemoglobin (Ox-Hb). One protease hydrolysed small fluorogenic substrates in addition to Ox-Hb. Its molecular mass was approximately 700 kDa and it consisted of several subunits ranging in size from 22 to 30 kDa. This enzyme may be related to the high-molecular-mass multicatalytic proteinase previously isolated from a variety of tissue and cell types. The other Ox-Hb-degrading activity had an apparent molecular mass of 400 kDa on gel filtration, a subunit size of 110 kDa and an isoelectric point between 4.5 and 5.0. This protease also hydrolysed the small polypeptides insulin and glucagon, as well as other large proteins such as lysozyme. Insulin blocked the degradation of Ox-Hb and Ox-Hb blocked the hydrolysis of insulin by the purified protease. Thiol reagents and metal chelators strongly inhibited the hydrolysis of both Ox-Hb and insulin, whereas inhibitors of serine, aspartic and thiol proteases had little effect. These properties suggest that the Ox-Hb-degrading activity purified from rabbit erythrocytes is the cytosolic insulin-degrading enzyme that is believed to play a role in the metabolism of insulin in several tissues. We propose that this enzyme may also function as a key component in a cytoplasmic degradative pathway responsible for removing proteins damaged by oxidants.
authors.affiliation: Department of Animal Sciences, Rutgers University, New Brunswick, NJ 08903.
authors.forename: J M
authors.initials: JM
authors.lastname: Fagan
authors.forename: L
authors.initials: L
authors.lastname: Waxman
mesh_terms: D000818:Animals; D002845:Chromatography / Q000379:methods; D010450:Endopeptidases / Q000097:blood* / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D006455:Hemoglobins, Abnormal / Q000737:chemistry / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D006455:Hemoglobins, Abnormal; D007328:Insulin; D010659:Phenylhydrazines; D011480:Protease Inhibitors; C030299:phenylhydrazine; D010450:Endopeptidases