Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver.
An NADPH-dependent reductase, first shown in the 1930s to catalyze the methylene blue-dependent reduction of methemoglobin in erythrocytes, has now been characterized as a high-affinity heme-binding protein and has been detected in liver. Highly purified bovine erythrocyte reductase binds protohemin to form a 1:1 complex with a Kd of 7 nM. Binding of protohemin completely inhibits reductase activity. Other tetrapyrroles and fatty acids also bind to the reductase and inhibit its activity. Protoporphyrin, hematoporphyrin, and coproporphyrin form 1:1 complexes with Kd values ranging from 1 to 5 microM. The inhibition constants for a number of saturated and unsaturated fatty acids range from 6 to 52 microM. A protein that is immunologically cross-reactive to the reductase has been detected in the cytosolic fractions of bovine and rat liver and of bovine, rat, rabbit, and human erythrocytes. By immunoblot analysis, the bovine liver and erythrocyte proteins appear identical in size, as do the rat liver and erythrocyte proteins. The concentration of the protein in bovine erythrocytes has been estimated by quantitative immunoblotting to be 10 microM. The detection of this protein in liver cells, the demonstration of its binding properties, and its weak reductase activity bring into question the long-held belief that this is uniquely an erythrocyte protein and that it functions as a reductase.
Xu
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Quandt
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Hultquist
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D000818:Animals; D002352:Carrier Proteins / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D042966:Cytochrome-B(5) Reductase / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D009249:NADP; D042966:Cytochrome-B(5) Reductase