Studies on methemoglobin reductase: Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes

Abstract

An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b 5 reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b 5 reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b 5 to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b 5 from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b 5 during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b 5 stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b 5 and methemoglobin. An antibody to purified, trypsin-solubilized cytochrome b 5 from rat liver microsomes was shown to inhibit erythrocyte cytochrome b 5 reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b 5 preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b 5 of human erythrocytes are immunochemically similar to NADH-cytochrome b 5 reductase and cytochrome b 5 of liver microsomes, respectively.