Studies on a fluorescent insect growth regulator metabolite complex with house-fly microsomal cytochrome P-450

The fluorescent insect growth regulator 55-(dimethylamino)-1-naphthalenyl]amino]-1,3-benzodioxole (DNSAB) forms a metabolite complex with house-fly microsomal cytochrome P-450. Formation of the metabolite complex is dependent on the presence of NADPH and O₂; NADH supports the reaction at a reduced rate. The presence of antibodies to house-fly cytochrome c (P-450) reductase in reaction mixtures inhibits the complex formation, indicating that the reductase is necessary for transfer of electrons from NADPH to cytochrome P-450 to complete the reaction. In the oxidized form, the metabolite complex has a single absorbance maximum at 431 nm, whereas the reduced form has two absorbance maxima at 426 (major) and 455 nm (minor). The pH of the media affects the extinction of the 426- and 455-nm Soret bands; increased pH decreases the extinction of the 426-nm band and increases the extinction of 455-nm band. Formation of the DNSAB metabolite-cytochrome P-450 complex decreases the amount of CO-reactive cytochrome P-450 by 24%. The metabolite complex is not dissociable by treatment with ferricyanide or by using centrifugation techniques. Dissociation is accomplished by addition of DNSAB to the oxidized metabolite complex. Kinetic analysis of the complex formation gives apparent K_m and V_max values at 2.55 ± 1.0 μM and 1.1 ± 0.4 × 10^?2 ΔA min^?1 nmol^?1 cytochrome P-450, respectively. Addition of juvenile hormone (E,E)-cis-methyl-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate; JH] to the reaction medium competitively inhibits the formation of the metabolite complex giving an inhibition constant of 16 μM. DNSAB synergized the lethal effects of JH against Aedes aegypti larvae threefold; however, JH did not synergize DNSAB. These data suggest that DNSAB may acquire its hormonal qualities by complexing a species of cytochrome P-450 that metabolizes JH, thereby prolonging the in vivo lifetime of this hormone.