Cytochrome P3-450 cDNA encodes aflatoxin B1-4-hydroxylase

Aflatoxin B1 (AFB1), a potent hepatocarcinogen and ubiquitous dietary contaminant in some countries, is detoxified to aflatoxin M1 (AFM1) via cytochrome P-450-mediated AFB1-4-hydroxylase. Genetic studies in mice have demonstrated that the expression of AFB1-4-hydroxylase is regulated by the aryl hydrocarbon locus and suggested that different cytochrome P-450 isozymes catalyze AFB1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. We have now examined lysates from mammalian cells infected with recombinant vaccinia viruses containing expressible cytochrome P1-450 or P3-450 cDNAs for their ability to metabolize AFB1 to AFM1. Our results show that cytochrome P3-450 cDNA specifies AFB1-4-hydroxylase. This is the first direct assignment of a specific cytochrome P-450 to an AFB1 detoxification pathway. This finding may have relevance to the dietary modulation of AFB1 hepatocarcinogenesis.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Mutagenic activation of aflatoxin B1 by P-450 HFLa in human fetal livers

The genotoxic and mutagenic activation of promutagens by human fetal livers was measured by the induction of umu gene expression in Salmonella typhimurium TA1535/pSk1002. Liver homogenates of human fetuses were the most active for the mutagenic activation of aflatoxin B1 (AFB1), followed by 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), and to a lesser extent by 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1). The amounts of P-450 HFLa immunochemically determined in human fetal livers correlated highly with the induction of umu gene expression by AFB1 (r = 0.98, n = 5). P-450 HFLa catalyzed the mutagenic activation of AFB1 in a reconstituted system: cytochrome b5 markedly stimulated the activation. Anti-P-450 HFLa antibodies inhibited the mutagenic activation of AFB1 in a dose-dependent manner. These results strongly support the idea that P-450 HFLa is responsible for the mutagenic activation of AFB1 in human fetal livers.     

Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers.

Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501A1 but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2B1. In addition, P-450HFLb also cross-reacted with both monospecific antibodies to rat CYP1A1 and CYP1A2. However, P-450HFLb was shown to be an immunochemically distinct form of cytochrome P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to be individual differences in the amounts of P-450HFLb expressed in fetal livers. The formation of mutagens from IQ (but not from AFB1) in fetal liver homogenates was inhibited by the antibodies to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagenic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Complete cDNA sequence of a major 3-methylcholanthrene-inducible cytochrome P-450 isozyme (P-450AFB) of Syrian hamsters with high activity toward aflatoxin B1

Cytochrome P-450AFB is major isozyme inducible by 3-methylcholanthrene in Syrian golden hamsters and shows high potency toward aflatoxin B1 activation. We have isolated and sequenced cDNA clones to P-450AFB by immunoscreening a hamster liver cDNA library in lambda gt11. The longest clone contains an open reading frame of 1482 nucleotides and encodes a protein of 494 amino acids with a molecular weight of 57,420. The sequence of P-450AFB shares a 73% and 65% homology with that of mouse P-450 15 alpha (IIA3) and rat P-450a (IIA1), respectively, indicating that P-450AFB is a unique gene of the P-450IIA subfamily. The apparent concentration of a mRNA species hybridizable to the clone as well as the concentration of a protein immunoreactive to P-450AFB was increased significantly by the treatment with 3-methyl-cholanthrene, which indicates that the increase in P-450AFB protein is due mainly to an elevation of the mRNA.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes.

Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D₃ when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D₃ metabolism.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Electron transport in cytochromes P-450 by covalent switching.

The mechanism of electron transfer in cytochrome P-450cam is presented in terms of a covalent switching mechanism. We present a model of putidaredoxin built by homology, which helps explain protein-protein interactions. The mechanism is general enough to account for the genetic variations found in the superfamily of cytochromes P-450. The detail should assist in the design of novel P-450 inhibitors and may have wider implications. The sequence analysis supports our protein model, and highlights the role of cystein and aromatic residues in electron-transport mechanisms. Eukaryotic cytochromes P-450 appear to have evolved their own intramolecular tryptophan electron-transfer mediator, unlike prokaryotic P. putida P-450cam, which still relies upon the C-terminal tryptophan of its attendant electron-transport protein, putidaredoxin. On this basis our protein model is capable of rationalizing the transfer of electrons from NADH to the active site of P-450. At the electronic level the covalent switching that transfers pairs of electrons not only provides a plausible mechanism, but may also have ramifications in a wider context.     

Modified acetylenic steroids as potent mechanism-based inhibitors of cytochrome P-450SCC

Synthesized 20-(4-tetrahydropyranyl-1-butynyloxy)-5-pregnen-3 alpha,20 beta- diol [steroid I] and 20-(3-tetrahydropyranyl-1-propargyloxy)-5-pregnen- 3 alpha,20 beta-diol [steroid III] have been found to inactivate purified adrenocortical cytochrome P-450SCC. When incubated with the enzyme under turnover conditions, steroid I inactivated cytochrome P-450SCC by about 85% in 40 min. This is in contrast to the free triol analog, steroid II which inactivated the enzyme by only 45% within the same incubation period. A comparison of steroid III with its free triol analog, steroid IV, also showed that the diol is a more effective inactivator of the enzyme than the triol. The partition ratio was calculated by two different methods. Each of the steroids I-IV bound to the enzyme with spectrophotometric dissociation constant (Ks) in the micromolar range, producing Type II low spin spectra changes during titration of the enzyme. In addition, it was found that the binding of each of the compounds to the enzyme occurred without inactivation of the enzyme and that the inactivation under turnover condition, is not as a result of conversion to the denatured P-420 species. This demonstrated that steroids I and III could correctly be designated as mechanism-based (suicide) inhibitors. The kinetic studies demonstrated that steroids with the tetrahydropyranyl substituent are more potent inhibitors of cytochrome P-450SCC as shown by an initial turnover rate of 0.06 min-1, an inactivation rate constant of 0.05 min-1, and a partition ratio of about 1.0 for steroid I. Based on our finding, possible mechanisms of inactivation of cytochrome P-450SCC by these acetylenic steroids are proposed.     

Role of cytochrome P-450IIE1 in N-nitroso-N-methylaniline induced hepatocyte cytotoxicity.

1. The cytotoxicity of N-nitrosomethylaniline (NMA) towards hepatocytes isolated from rats was prevented by acetone or ethanol (inhibitors for cytochrome P-450IIE1) but not by metyrapone or SKF525A (inhibitors for cytochrome P-450IIB1/2). Various alcohols, secondary ketones and isothiocyanates that induced cytochrome P-450IIE1 were also found to be protective. Various aromatic and chlorinated hydrocarbon solvents that are substrates or inducers of cytochrome P-450IIE1 also prevented NMA cytotoxicity. Nitrogen-containing heterocycles that induced cytochrome P-450IIE1 were less effective. Further evidence that cytochrome P-450IIE1 was responsible for the activation of NMA was the marked increase in hepatocyte susceptibility if hepatocytes from pyrazole-induced rats were used. 2. NMA was more cytotoxic to hepatocytes isolated from phenobarbital-pretreated rats than uninduced rats. However, metyrapone now prevented and SKF525A delayed the cytotoxicity whereas ethanol, acetone, allyl isocyanate, isoniazid or trichloroethylene had no effect on the susceptibility of phenobarbital-induced hepatocytes. Furthermore, microsomes isolated from phenobarbital-pretreated rats had higher NMA-N-demethylase activity which was more inhibited by metyrapone and SKF525A than that of uninduced microsomal activity. By contrast the N-demethylase activity of phenobarbital induced microsomes was more resistant to acetone, ethanol, hexanal, trichloroethylene and toluene than uninduced microsome. 3. The above results suggest that cytochrome P-450IIE1 catalyses the cytotoxic activation of NMA in normal or pyrazole-induced hepatocytes whereas cytochrome P-450IIB1/2 is responsible for cytotoxicity in phenobarbital-induced hepatocytes.     

On the role of phospholipids in the reconstituted cytochrome P-450 system. A model study using dilauroyl and distearoyl glycerophosphocholine

The difference in pentoxyresorufin O-dealkylating activity observed in a reconstituted system containing dilauroylglycerophosphocholine (Lau2GroPCho) or distearoylglycerophosphocholine (Ste2GroPCho) was used as a model to study the role of phospholipids in the reconstituted cytochrome P-450b (IIB1) system. The hypotheses proposed in the literature for the role of phospholipids in the reconstituted cytochrome P-450 system, mainly based on the comparison of systems without phospholipid and with Lau2GroPCho, were either validated or shown to be unlikely when tested by comparing reconstituted systems with different phosphatidylcholines. The higher activity in the Lau2GroPCho system as compared to the Ste2GroPCho system cannot be ascribed to (a) an increased affinity of cytochrome P-450 for the NADPH-cytochrome reductase in the Lau2GroPCho system, also not to (b) a Lau2GroPCho-dependent dissociation of protein multimers, nor to (c) a change in the spin state of the heme. We found a different apparent Km for pentoxyresorufin in the Lau2GroPCho system compared with the Ste2GroPCho system. Furthermore, we found a difference between the cytochrome P-450b tryptophan fluorescence polarization of the Lau2GroPCho system and the Ste2GroPCho system as well as with a system without phosphatidylcholine. From these observations it is concluded that the higher activity of the Lau2GroPCho system compared with the Ste2GroPCho system or with a system without additional phosphatidylcholine may at least in part be caused by a difference in the conformation of the cytochrome P-450 molecules in these systems. Furthermore, the different effects of both phosphatidylcholines on the Km and V for pentoxyresorufin not only suggest a role of phospholipids in the binding of the substrate to the active site of the cytochrome P-450 molecule, but also on the efficiency of electron transfer from NADPH-cytochrome reductase to cytochrome P-450.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of hepatic microsomal cytochromes P-450 from beta-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with beta-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with beta-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (Mr 55000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identity with cod cytochrome P-450b (Mr 54000), possibly as a result of contaminating cytochrome P-450c in this fraction.     

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.     

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.     

Cytochrome P-450 and halothane metabolism. Decrease in rat liver microsomal P-450 in vitro

Anaerobic in vitro incubation of microsomes from phenobarbital(PB)-induced rats with halothane results in an irreversible decrease of measurable cytochrome P-450. There is a parallel decrease in heme content under the same incubation conditions. However, microsomes from 3-methylcholanthrene(3-MC)-induced or untreated animals do not show a reduction in cytochrome P-450 content. Aerobic incubation with halothane results in a decrease of cytochrome P-450 which can be completely reversed by dialysis or the addition of potassium ferricyanide. These latter treatments only partially restore the cytochrome P-450 levels following anaerobic incubations. The decrease in cytochrome caused by halothane is not associated with measureable heme N-alkyl adduct formation; lipid peroxidation does not play a role as indicated by the lack of effect of 1 mM EDTA or a decrease in glucose-6-phosphatase activity. Halothane metabolites are bound irreversibly to microsomal protein as determined by gel electrophoresis only when the oxygen concentration is very low. The mechanism of cytochrome P-450 decrease is consistent with the formation of a reactive metabolite which binds to the protein portion and also destroys heme.