Insect Cytochrome P450 Research Articles

Cytochrome P-450 in insects—V.: Monoterpene induction of cytochrome P-450 and associated monooxygenase activities in the larva of the variegated cutworm Peridroma saucia (Hübner)

1. The induction of microsomal cytochrome P-450 and associated polysubstrate monoxygenase activities by natural levels (0.01–0.1%) of the naturally occurring monoterpenes pulegone, menthone and menthol was examined in sixth-instar larvae of the variegated cutworm ( Peridroma saucia Hübner). 2. The monoterpenes increased cytochrome P-450 levels, aldrin and heptachlor epoxidase activity in microsomes from midgut and other tissues of the midsection. 3. NADPH-dependent cytochrome c reductase levels generally were not increased in midgut microsomes and were increased in carcass microsomes by higher doses of the inducers. 4. Induction by any of the monoterpenes increased the size of the Type I (substrate) binding spectra obtained with pulegone, menthone and nerolidol, but not those obtained with menthol. 5. The induction of polysubstrate monooxygenases by these compounds may increase the resistance of the larvae to both host plant allelochemicals and to insecticides in the field.

Cytochrome P-450 in insects : 3. Increase in substrate binding by microsomes from phenobarbital-induced house flies

Microsomal membranes prepared from the abdomens of two insecticide-susceptible house-fly strains (NAIDM and CSMA) and one insecticide-resistant strain (Rutgers) were examined for evidence of substrate binding by cytochrome P-450. The substrates studied in detail were benzphetamine, nerolidol, and menthone, and to a lesser extent, aldrin and heptachlor. Contrayy to previous reports, type I spectra were obtained with cytochrome P-450 from the susceptible flies. As expected, Rutgers strain P-450 also exhibited type I spectra. The spectra were typical in all cases but one (nerolidol) and were about one-third to one-seventh the size of those obtained with microsomal P-450 from the Rutgers strain. With the exception of those obtained with menthone, substrate-induced spectra were greatly enhanced (about two- to four-fold) with the P-450 from susceptible flies treated with phenobarbital. This treatment did not increase substrate binding by the P-450 from Rutgers flies. In a comparison of the epoxidation of aldrin and heptachlor by induced and noninduced microsomes, it was noted that only the heptachlor epoxidase was increased in all strains. These studies indicate that phenobarbital induction of susceptible flies results in the production of cytochrome P-450 with spectral and enzymatic properties resembling those of resistant flies.

Cytochrome P-450 in insects : 1. Differences in the forms present in insecticide resistant and susceptible house flies

Soluble cytochrome P-450 prepared from the microsomal fraction of abdomen homogenates of an insecticide resistant strain (Rutgers) and a susceptible strain (NAIDM) of the house fly, Musca domestica L., was characterized by spectral and electrophoretic methods. Six chromatographically distinct fractions were obtained after chromatography on DEAE-cellulose and hydroxylapatite. Examination of the six fractions by difference spectrophotometry indicated that the wave lengths for maximum absorption of the cytochrome P-450-carbon monoxide complexes were at 450, 451, and 452 nm for the NAIDM fractions and at 449, 450, and 451 nm for the Rutgers fractions. The type II binding spectra of the cytochrome P-450 in each fraction were measured with n-octylamine. Several of these resembled spectra which, in studies of hepatic cytochrome P-450, have been shown to be due to the presence of the high spin form of this hemoprotein. Four of the fractions from the resistant strain were of this type compared to one from the susceptible strain. Electrophoresis experiments indicated that there were at least three hemoproteins in the 40,000–60,000 molecular weight range in the fractions from the resistant strain while four could be detected in those from the susceptible strain. The specific aldrin epoxidase activity of the most active Rutgers fractions was considerably higher than that of similar fractions from the NAIDM microsomes in reconstitution experiments.

Ecdysone 20-monooxygenase: Characterization of an insect cytochrome P-450 dependent steroid hydroxylase

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworn, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrosegradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg 2+ and exhibits pH and temperature optima at 7.5 and 30°C, respectively. The enzyme complex has an apparent K m for ecdysone of 1.60 × 10 −7 M and is competitively inhibited by its product, ecdysterone, with an apparent K i of 2.72 × 10 −5 M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.

Insect cytochrome P-450.

Two approaches may be used to study the function of cytochrome P-450 in insects: (a) an evaluation of the spectral and catalytic properties of the hemoprotein while associated with microsomal membranes; (b) the solubilization, resolution and purification of the microsomal mixed-function oxidase system. The first approach has provided some understanding of the biochemical factors involved in the metabolism of a variety of compounds, including pesticides, drugs, hormones and many other xenobiotics. However, solubilization of the monooxygenase system allows the study of each of its components individually, providing a better insight on the sequence of events leading to the hydroxylation of a substrate, the type of intermediates formed, and the rate-limiting step(s). This report discusses studies carried out with the monooxygenase system associated with microsomal membranes, as well as procedures to solubilize and partially purify its components from housefly microsomes. The latter involves solubilization with either Triton X-100 or sodium cholate, followed by either ammonium sulfate fractionation, Sephadex G-200, DEAE-Sephadex A-50 column chromatography or by omega-amino-n-octyl-Sepharose 4B affinity chromatography. These procedures have shown that two cytochrome P-450 species (P-450 and P-450I) are present in microsomes isolated from a resistant housefly strain. Induction with either naphthalene or phenobarbital appears to increase cytochrome P-450I preferentially.