Purification and characterization of cytochrome b5 reductase from the house fly, Musca domestica.
NADH-cytochrome b5 reductase (b5R) from the house fly was purified through solubilization of microsomes with Triton X-100 followed by DEAE, carboxylmethyl and 5'-ADP affinity column chromatography. Yields of 9% with a 320-fold increase in NADH-ferricyanide reductase specific activity and 2% with a 76-fold increase in NADH-cytochrome b5R specific activity were obtained. Two forms of b5R, a major form with the apparent molecular mass of 31 kDa and a minor form of 33 kDa, were obtained. Both forms of purified b5R could reduce cytochrome b5 and both could use NADH or NADPH as an electron donor, although NADH was more efficient. Kinetics of the b5R activities were also studied. The 31-kDa b5R consists of integral of 291 amino acids with the NH2-terminal sequence of Thr-Ala-Arg-Leu-Arg-Thr-Leu-Ile-Asp-Ala. An antiserum developed against the 31-kDa b5R recognized both forms of b5R. Using this polyclonal antiserum as a probe, immunologically reactive proteins were found in microsomes from five species of Diptera, mouse and rat liver but not in spider mites nor insects from other orders.
Zhang
M
M
Department of Entomology, Cornell University, Ithaca, NY 14853-0999, USA.
Scott
J G
JG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D003579:Cytochrome Reductases / Q000302:isolation & purification*; D042966:Cytochrome-B(5) Reductase; D005292:Ferricyanides; D006793:Houseflies / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction
D000596:Amino Acids; D005292:Ferricyanides; C007931:hexacyanoferrate III; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase