On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.     

Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

Effect of spermine on the inhibition by fatty acid on AMP deaminase reaction as a control system of the adenylate energy charge in yeast

Treatment of rats with pyrazole elevated the hepatic microsomal dimethylnitrosamine demethylase activity (DMNd) by several fold. Methylethylnitrosamine demethylase activity was also increased by pyrazole, but some classical monooxygenase activities were not induced. The treatment induced a new protein species which has an apparent molecular weight of 52,000 dal and is believed to be a cytochrome P-450 isozyme. The involvement of a hemoprotein in the pyrazole-induced DMNd was demonstrated in an experiment with CoCl₂ which decreased both the microsomal cytochrome P-450 content and DMNd. The induced enzyme with a single K_m value of 0.061 mM and V_max of 12.1 nmol/min/mg is probably the most efficient enzyme known to metabolize nitrosamines. NADPH-cytochrome P-450 reductase was also demonstrated to be an essential component enzyme of the DMNd. These results further substantiate the idea that the P-450-containing monooxygenase is responsible for the metabolism of dimethylnitrosamine in both the control and pyrazole induced microsomes.     

Modulation of ethylene synthesis in acotyledonous soybean and wheat seedlings

The characteristics of ethylene production and ACC conversion in 8-day-old soybean seedlings were examined and a relationship between cytochrome P-450 activity and ethylene-forming enzyme (EFE) activity was found. An atmosphere containing 10% carbon monoxide (CO) significantly inhibited ethylene production and ACC conversion in control soybean seedlings, but had only a slight effect on soybean seedlings treated with uniconazole. Foliar application of triclopyr, a pyridine analogue of the phenoxy herbicides, significantly increased ethylene production and ACC conversion in control, but not in uniconazoletreated seedlings. Triclopyr treatment also resulted in a three-fold increase in extractable cytochrome P-450 of 5-day-old etiolated soybeans. At equimolar concentrations tetcyclacis was more effective than uniconazole in reducing shoot elongation and endogenous ethylene production. Although uniconazole and tetcyclacis did not inhibit ACC conversion in nonherbicide-treated soybean seedlings, they did prevent the observed increase in ACC-dependent EFE activity following triclopyr application. However, the rate of ACC conversion in etiolated soybean segments was sensitive to uniconazole, and tetcyclacis inhibited the rate of ACC conversion by 2.6-fold in etiolated soybean segments within 4 h after treatment. Microsomal membranes were isolated from 5-day-old naphthalic anhydride-treated etiolated wheat shoots as this tissue contains much higher cytochrome P-450 levels than soybean shoots. Optical difference spectroscopy demonstrated that ACC generated binding spectrum characteristic of a reverse-type-I cytochrome P-450 substrate when combined with reduced microsomes. In vitro conversion of ACC to ethylene by microsomal membranes was NADPH-dependent, inhibited by CO, and had an apparent K_m and V_max of 45 M and 0.345 nl/mg protein/h, respectively. These results suggest that cytochrome P-450-mediated monooxygenase reactions may be intimately involved in the conversion of ACC to ethylene in young soybean and wheat seedlings.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

Microsomal cytochromes of Candida tropicalis grown on alkanes

A comparison of methods used in isolating microsomes and in measuring microsomal cytochrome P-450 demonstrated that separation following protoplast lysis gave the best results. By this latter technique a high amount of cytochrome P-450 (0.2–0.3 nmol/mg) was recovered but cytochrome P-420, considered as the denatured form, was absent.The alkanes specifically induce cytochromes P-450 and b₅ localized on the microsomes. The denaturation in vivo of cytochrome P-450 into cytochrome P-420 even occurs during storage at 1 °C. This degradation is increased during preparation of subcellular fractions if no preventive measures are taken.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.     

Cytochrome P-450 and hydroxylating activity of microsomal preparations from whole houseflies

1. A new microsomal preparation, obtained from whole houseflies is described in terms of its cytochrome P-450 content and its hydroxylating activity. 2. Microsomes prepared from whole-fly brei, obtained with the aid of a mortar (a procedure that avoids the destruction of sarcosomes), contain 0.265nmol of cytochrome P-450 and hydroxylate naphthalene at a rate of 28.5nmol/mg of microsomal protein in 30min at 30 degrees C. This corresponds to 104nmol of naphthalene hydroxylated/nmol of cytochrome P-450. This is the highest rate ever reported for housefly and rat liver microsomal preparations. 3. Microsomal fractions prepared by procedures that do not retain the integrity of sarcosomes show the presence in the CO-difference spectrum of a 428nm peak. This cytochrome is associated with sarcosomal microsomes and it may be involved in the inhibition of insect microsomal mixed-function oxidases, although other factors cannot be discarded at present. 4. The inability to show cytochrome P-450 in microsomal fractions isolated from whole houseflies by other procedures may be at least partially due to a masking effect brought about by contamination with the sarcosomal cytochrome.     

Drug metabolism in rat colon: Resolution of enzymatic constituents and characterization of activity

A direct demonstration of the basis of mixed function oxidase activity in rat colonic mucosa was achieved by resolution of microsomes into two components, cytochrome P-450 and cytochrome P-450 reductase, which on recombination with phosphatidylcholine catalyzed hydroxylation of benzo[]pyrene and benzphetamine. Reconstitution of hydroxylation activity requires both the cytochrome P-450 component and the cytochrome P-450 reductase component in addition to phospholipid. Omission of either of the protein components or the phospholipid component reduces the activity almost to background levels. The kinetic parameters (K_m values) for the reconstituted system suggest that the colonic mucosal system is quite similar to the liver microsomal system in its catalytic capacity as well as in its enzymic composition. The purified colon components substitute for their liver counterparts reasonably well, again consistent with the argument that the colon mucosal mixed function oxidase system is analogous to the liver system.     

The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase

Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.     

Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly

Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-³H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O₂ for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg^?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450.     

Induction of cytochrome P-450 and P-448 in the outer membrane of mouse liver nuclei by phenobarbital and benzo [α-]pyrene

This investigation confirms the presence of the inducible mixed function hydroxylase enzyme system in nuclear membranes. The cytochrome P-450 spectrum and demethylase activity, markers of the enzyme system, were used to define its localization to the outer membrane envelope. Intact BALB/c mouse liver nuclei isolated and purified in Mg⁺⁺ sucrose media of low ionic strength gave CO-dithionite reduced difference spectra of cytochrome P-450 and P-448. Phenobarbital induced P-450 by 40% while the carcinogenic hydrocarbon, benzo [α] pyrene, induced P-448 twofold. A corresponding increase was also observed in the microsomes of the same tissue preparations. No microsomal contamination of nuclear preparations was found. Intact nuclei stripped of their outer membrane by 0.5% Triton X-100 treatment resulted in a striking absence of the P-450 which, however, was found to be present in isolated outer nuclear membranes.     

Developmental changes in rabbit pulmonary cytochrome P-450 subpopulations

Of the two characterized cytochrome P-450 subpopulations present in adult lung microsomes, only one (P-450_II) appears to be present in the neonate. Both this subpopulation and a second subpopulation (P-450_I) gradually increase over a period of several months, and account for most of the increase in lung cytochrome P-450 concentration during maturation. A third fraction of the cytochrome P-450, which is incapable of forming metabolic-intermediate complexes remains constant in concentration during maturation, thus decreasing from 60% of the total in the neonate to 20% in the adult. Metyrapone binding to lung cytochrome P-450 which increases during development does not correlate quantitatively with either of the two characterized subpopulations.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Platelet cytochrome P-450: A possible role in arachidonate-induced aggregation

Platelet microsomes were shown to contain cytochromes P-450 and b₅ and their respective reductases, NADPH-cytochrome c reductase and NADH-cytochrome b₅ reductase. Metyrapone and carbon monoxide (CO), two inhibitors of cytochrome P-450, inhibited both the arachidonic acid-induced platelet aggregation and the formation of aggregating factors from arachidonic acid by isolated microsomes. In addition metyrapone produced a type II spectral change with platelet microsomal cytochrome P-450. The data suggest that cytochrome P-450 may play a role in the complex enzyme systems which convert arachidonic acid to the platelet aggregating factors, cyclic endoperoxides and thromboxane A₂.     

The cytochrome P450-containing monooxygenase of Trichosporon cutaneum: Occurrence and properties

Trichosporon cutaneum metabolizes glucose purely oxidatively and cytochrome P450 was not detected in the reduced CO-difference spectrum of whole cells. However, in the isolated microsomal fraction the corresponding monooxygenase was present as shown by the appearence of cytochrome P450, NADPH-cytochrome c (P450) reductase and cytochrome b₅. The absorption maximum of the terminal oxidase in the reduced CO-difference spectrum shifted between 447 and 448 nm. Derepression of biosynthesis of all components was achieved by transition of the cells from carbon- to oxygen-limited growth in continuous culture. The monooxygenase exhibited aminopyrine demethylation activity but not -hydroxylation activity of lauric acid. With respect to the growth limiting nutrient (carbon and oxygen respectively), mitochondrial cytochrome content showed an analogous behavior as cytochrome P450 and cytochrome b₅.