PURIFICATION AND CHARACTERIZATION OF CYTOCHROME-B(5) REDUCTASE FROM THE HOUSE-FLY, MUSCA-DOMESTICA

NADH-cytochrome b(5) reductase (b5R) from the house fly was purified t hrough 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 ac tivity and 2% with a 76-fold increase in NADH-cytochrome b5R specific activity were obtained. Two forms of b5R, a major firm with the appare nt molecular mass of 31 kDa and a minor form of 33 kDa, were obtained. Both forms of purified b5R could reduce cytochrome b(5) and both coul d use NADH or NADPH as an electron donor, although NADH was more effic ient. Kinetics of the b5R activities were also studied. The 31-kDa b5R consists of similar to 291 amino acids with the NH2-terminal sequence of Thr-Ala-Arg-Leu-Arg-Thr-Leu-Ile-Asp-Ala. An antiserum developed ag ainst the 31-kDa b5R recognized both forms of b5R. Using this polyclon al antiserum as a probe, immunologically reactive proteins were found in microsomes from five species of Diptera, mouse and rat liver but nu t in spider mites nor insects from other orders.