Stimulation of cytochrome P450 reactions by apo-cytochrome b5: evidence against transfer of heme from cytochrome P450 3A4 to apo-cytochrome b5 or heme oxygenase.

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b(5) (b(5)). Two major explanations are (i) direct electron transfer from b(5) and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b(5) or b(5) devoid of heme (apo-b(5)), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b(5) but not by apo-b(5). Recently, a proposal has been made by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b(5) can be explained only by transfer of heme from P450 preparations to apo-b(5), enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6beta-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only approximately 5% of the heme can be transferred to apo-b(5), which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally, b(5) supported NADH-b(5) reductase/P450 3A4-dependent testosterone 6beta-hydroxylation, but apo-b(5) did not. Thus, apo-b(5) can stimulate P450 3A4 reactions as well as b(5) in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b(5) cannot be used to explain the catalytic stimulation.     

Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene

A novel P450 cDNA was isolated from a rat lung lambda gt11 library by hybridization with the rat P450 IIB1 cDNA probe. The cDNA-deduced amino acid sequence of this clone was 71% and 73% similar to rat IIA1 and IIA2 P450s; it was, therefore, designated IIA3 as the third member of the rat IIA subfamily. IIA3 demonstrates only 55% amino acid similarity with IIB1. Interestingly, this P450 also shared 85% and 94% amino acid similarities with human IIA3 and a mouse testosterone 15 alpha-hydroxylase P450, respectively, indicating that these P450s are orthologous counterparts to rat IIA3. Chromosome mapping, using mouse-hamster somatic cell hybrids, revealed that the IIA3 gene is localized on mouse chromosome 7. The IIA3 mRNA was detected in rat lung, and its level was induced 3-fold by treatment of rats with 3-methylcholanthrene. No IIA3 mRNA was seen in the liver, kidney, or intestine, even after long exposure of Northern blot filters to X-ray film. In contrast, the orthologous mouse and human IIA3 genes are expressed in liver.     

Cellular localization of cytochrome P450IIA1 in testes of mature Sprague-Dawley rats.

Previous studies have shown that a prominent site of extrahepatic cytochrome P450IIA1 in male rats is the testis. We investigated the cellular location of cytochrome P450IIA1 in the testes of adult rats. Using specific isolation of testicular compartments and individual cell types, as well as in vivo removal of Leydig cells by ethane dimethyl sulfonate, we determined the cellular location of cytochrome P450IIA1 using testosterone hydroxylation assay, Western immunoblotting, and immunohistochemical analysis. Enriched Leydig cell fractions had the greatest testosterone 7 alpha-hydroxylase activity as well as immunoreactivity. Immunohistochemical analysis confirmed that the cellular location of cytochrome P450IIA1 was specific to Leydig cells. The specific localization of enzyme systems that are involved in xenobiotic activation may have important implications for inducing specific cell toxicity by compounds that exert their effects in the testes.     

Age-dependent expression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Age-dependent exression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2α-, 2β-, 15α-, 16α-, and 16β-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7α-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible form) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6β-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Gene conversion and differential regulation in the rat P-450 IIA gene subfamily. Purification, catalytic activity, cDNA and deduced amino acid sequence, and regulation of an adult male-specific hepatic testosterone 15 alpha-hydroxylase

Previous studies on regulation of the rat hepatic P-450 IIA1 cDNA have provided evidence for a second gene closely related to but regulated in a manner quite distinct from P-450 IIA1. Experiments were carried out to isolate the cDNA for this second P-450 gene, designated IIA2, in order to study more directly its regulation and relationship to IIA1. A full length cDNA to IIA2 was isolated from an adult male rat liver lambda gt11 library and sequenced completely. The IIA2 cDNA shared 93% nucleotide and 88% deduced amino acid similarities with the previously characterized IIA1 cDNA (Nagata, K., Matsunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 2787-2793). The protein, deduced from the cDNA, contained 492 amino acids and a calculated Mr of 56,352. Comparison of the IIA1 and IIA2 cDNAs revealed areas of low nucleotide similarity interspersed with areas of absolute identity, suggesting that gene conversions have played a role in the evolution of the IIA subfamily. Expression of IIA1 and IIA2 mRNAs in rat liver during development was studied with use of specific oligonucleotide probes. IIA1 mRNA was increased within 1 week after birth in both male and female rats; however, its postpubertal expression was decreased in males yet remained elevated in females. In contrast, IIA2 mRNA was markedly induced in male rat liver at puberty but was not detectable in females at any age examined. Furthermore, only IIA1 mRNA was induced by treatment of rats with 3-methylcholanthrene. Although IIA1 and IIA2 mRNAs were actively expressed in hepatic tissue, no evidence for their expression was found in lung, kidney, or intestine, suggesting that the IIA genes have tissue-specific promoters. Reconstituted enzyme assays on the purified protein products P-450 IIA1 and P-450 IIA2 showed that, although both enzymes share considerable sequence similarity, their positional specificities toward the prototype substrate testosterone are strikingly different.     

Conformational dynamics and molecular interaction reactions of recombinant cytochrome p450scc (CYP11A1) detected by fluorescence energy transfer.

Bovine adrenocortical cytochrome P450scc (P450scc) was expressed in Escherichia coli and purified as the substrate bound, high-spin complex (16.7 nmol of heme per mg of protein, expression level in E. coli about 400-700 nmol/l). The recombinant protein was characterized by comparison with native P450scc purified from adrenal cortex mitochondria. To study the interaction of the electron transfer proteins during the functioning of the heme protein, recombinant P450scc was selectively modified with fluorescein isothiocyanate (FITC). The present paper shows that modified P450scc, purified by affinity chromatography using adrenodoxin-Sepharose to remove non-covalently bound FITC, retains the functional activity of the unmodified enzyme, including its ability to bind adrenodoxin. Based on the efficiency of resonance fluorescence energy transfer in the donor-acceptor pair, FITC-heme, we calculated the distance between Lys(338), selectively labeled with the dye, and the heme of P450scc. The intensity of fluorescence from the label dramatically changes during: (a) denaturation of P450scc; (b) changing the spin state or redox potential of the heme protein; (c) formation of the carbon monoxide complex of reduced P450scc; (d) as well as during reactions of intermolecular interactions, such as changes of the state of aggregation, complex formation with the substrate, binding to the electron transfer partner adrenodoxin, or insertion of the protein into an artificial phospholipid membrane. Selective chemical modification of P450scc with FITC proved to be a very useful method to study the dynamics of conformational changes of the recombinant heme protein. The data obtained indicate that functionally important conformational changes of P450scc are large-scale ones, i.e. they are not limited only to changes in the dynamics of the protein active center. The results of the present study also indicate that chemical modification of Lys(338) of bovine adrenocortical P450scc does not dramatically alter the activity of the heme protein, but does result in a decrease of protein stability.     

The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a lambda gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu160----His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a lambda max at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation (turnover number of 15 min-1) and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIA mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. In addition, kinetics of coumarin metabolism in two livers were monophasic over the substrate concentration tested. Km values obtained from human liver (2.3 microM) were similar to those obtained from lysates of hepatoma cells expressing IIA3 (3.6-7.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Apo-cytochrome b5 as an indicator of changes in heme accessability: preliminary studies with cytochrome P450 3A4.

Cytochrome P450s (P450 or CYP) are the largest family of hemeproteins yet characterized. X-ray crystallographic studies have shown that the heme of the P450 hemeproteins is buried in the interior of the protein molecule. Unexplored are answers to questions concerning the role of heme in the folding of newly synthesized apo-P450s and the factors that influence changes in heme accessibility following modification of the pattern of folding of the holo-P450s. We have carried out the present studies to measure changes in heme accessibility in P450s. This is an initial step to determining whether heme-binding confers structural and functional integrity and stability to a P450 molecule. Recently, we have shown that apo-high molecular weight cytochrome b5 (apo-HMWb5) is an efficient acceptor of heme when added to a preparation of purified recombinant CYP3A4. In the present work we have studied heme binding by apo-HMWb5 when mixed with a number of different hemeproteins (myoglobin, hemoglobin, catalase, CYP4A1, CYP101, and CYP3A4). These hemeproteins differ in the location of the heme (i.e., surface or internal) allowing one to study changes in structure as measured by the process of heme transfer from one protein to another. It was found that heme transfer to apo-HMWb5 occurs relatively rapidly from hemeproteins where the heme is located at or near the surface or when the hemeprotein is denatured. In contrast, heme transfer from P450s to apo-HMWb5 occurs only following modification of the P450 structure with chaotropic agents. An exception is CYP3A4 where a measurable amount of heme is transferred to apo-HMWb5 in the absence of denaturing agents. The preliminary results described here employs apo-HMWb5 as an indicator for assessing changes in heme-availability of P450s as the protein-folding of the molecule is altered.     

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.     

Baculovirus-mediated expression and functional characterization of human NADPH-P450 oxidoreductase.

Human NADPH-P450 oxidoreductase (OR) is an intrinsically membrane-bound flavoprotein that serves to transfer electrons from NADPH to cytochrome P450. OR is also involved in the metabolic activation of chemotherapeutic alkylating agents. The human OR cDNA was engineered into baculovirus and the recombinant virus was used to infect Spodoptera frugiperda (Sf9) cells. Approximately 3.3% of total protein of infected cells was human OR. The enzyme was purified by ion exchange and affinity chromatography to a specific activity of 20 units/mg protein. Baculovirus-expressed OR displayed an absolute spectrum typical of the protein purified from tissue sources. The purified enzyme was able to support P450 activity in a reconstituted lipid vesicle system where maximal P450 activity was achieved at an OR/P450 ratio of 2. When recombinant OR and P450 DNA-containing baculoviruses were used to coinfect Sf9 cells, the OR/P450 ratio needed to achieve half maximal P450 catalytic activity was less than 0.5. These studies demonstrate the utility of baculovirus to analyze the functional and structural relationship of OR and P450.     

Bioreactor systems in drug metabolism: synthesis of cytochrome P450-generated metabolites

In this communication, we report that suspension cultures of Sf21 insect cells, co-infected with baculovirus containing the cDNA for a single cytochrome P450 and NADPH-cytochrome P450 oxidoreductase, can be employed successfully as "bioreactors" for the synthesis of milligram quantities of cytochrome P450-generated metabolite(s). Three standard or probe substrates for the human P450s were chosen for the initial biosynthetic experiments: testosterone, diazepam, and diclofenac. Testosterone (100 microM, 2.88 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to 6beta-hydroxytestosterone (2.3 mg) and 15beta-hydroxytestosterone (0.18 mg). Diazepam (100 microM, 2.9 mg/100 ml), added to a 100-ml CYP3A4 bioreactor, was converted to temazepam (1.1 mg), N-demethyldiazepam (0.35 mg), and oxazepam (0.15 mg). Diclofenac (100 microM, 3.18 mg/100 ml), added to a 100-ml CYP2C9 bioreactor, was converted to 4'-hydroxydiclofenac (2.6 mg). Since the goal for the development of the bioreactors was to provide a platform for both the production and subsequent purification of milligram quantities of P450-generated metabolite(s), a second 100-ml CYP2C9 bioreactor was used for the large-scale production and subsequent purification of 4'-hydroxydiclofenac. After 55 h of incubation, 7.95 mg of diclofenac was converted to 4.35 mg of 4'-hydroxydiclofenac, while 3.55 mg of unchanged diclofenac remained in the bioreactor. Using a simple preparative HPLC method, approximately 2.2 mg of 4'-hydroxydiclofenac and 1.9 mg of diclofenac were recovered from this experiment (28% yield). These results indicate clearly that suspension cultures of Sf21 insect cells coexpressing a cytochrome P450 and NADPH-cytochrome P450 oxidoreductase can be used effectively as bioreactors for the production and subsequent purification of milligram quantities of P450-derived metabolite(s).     

Redox properties of cytochrome p450BM3 measured by direct methods.

Cytochrome p450BM3 is a self-sufficient fatty acid monooxygenase consisting of a diflavin (FAD/FMN) reductase domain and a heme domain fused together in a single polypeptide chain. The multidomain structure makes it an ideal model system for studying the mechanism of electron transfer and for understanding p450 systems in general. Here we report the redox properties of the cytochrome p450BM3 wild-type holoenzyme, and its isolated FAD reductase and p450 heme domains, when immobilized in a didodecyldimethylammonium bromide film cast on an edge-plane graphite electrode. The holoenzyme showed cyclic voltammetric peaks originating from both the flavin reductase domain and the FeIII/FeII redox couple contained in the heme domain, with formal potentials of -0.388 and -0.250 V with respect to a saturated calomel electrode, respectively. When measured in buffer solutions containing the holoenzyme or FAD-reductase domain, the reductase response could be maintained for several hours as a result of protein reorganization and refreshing at the didodecyldimethylammonium modified surface. When measured in buffer solution alone, the cyclic voltammetric peaks from the reductase domain rapidly diminished in favour of the heme response. Electron transfer from the electrode to the heme was measured directly and at a similarly fast rate (ks' = 221 s-1) to natural biological rates. The redox potential of the FeIII/FeII couple increased when carbon monoxide was bound to the reduced heme, but when in the presence of substrate(s) no shift in potential was observed. The reduced heme rapidly catalysed the reduction of oxygen to hydrogen peroxide.     

Human aromatase in high yield and purity by perfusion chromatography and its characterization by difference spectroscopy and mass spectrometry

Expression of human cytochrome P450 aromatase (CYP19A1, aromatase) was accomplished at a high level using a baculovirus expression system in an insect cell suspension culture. Using the relatively new chromatographic technique of perfusion chromatography, a very rapid procedure for purification of the protein from solubilized cells was developed. At extraordinary flow rates of between 3 and 9 column volumes per minute, all chromatographic procedures could be performed, including setup, equilibration, and column regeneration steps, in less than 2 h, not including brief dialysis periods. Total yields were 40-52% and resulted in preparations with specific content values of 17.1 nmol aromatase/mg protein. Final purified preparations showed virtually no typical P450 spectra under standard conditions, but displayed full activity with typical enzyme kinetic parameters. These unusual results suggest that standard methods of P450 measurement are inappropriate when applied to aromatase. The findings are fully consistent with those encountered previously for purified preparations from a human placental source and led us to a new aromatase quantification method based on ligand-induced difference spectroscopy. A new HPLC assay is described which rapidly separates heme and apoprotein while measuring total heme content. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was employed with both glycosylated and deglycosylated forms of the final purified product to confirm its identity as a glycosylated cytochrome P450.     

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.     

利用杆状病毒Bac-to-Bac系统在Sf9细胞中表达真菌细胞色素P450nor

利用BactoBac杆状病毒载体表达系统将真菌细胞色素P450nor基因克隆至转移载体pFastBac1中, 得到重组质粒pFastBacP450nor, 再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中发生转座作用, 得到含P450nor基因的重组穿梭载体rBacmid pAcP450nor。分离提取重组Bacmid DNA, 并转染培养的昆虫细胞Sf9, 得到重组病毒rAcp450nor。经酶切和PCR 鉴定, 细胞色素P450nor基因正确地插入到病毒基因组的多角体蛋白基因启动子下, SDSPAGE分析证明：表达蛋白的分子量为43kD左右。Western blotting分析结果表明：有一条特定的杂交带存在, 且分子量相同（约43kD）。进一步证明了含有真菌细胞色素P450nor基因的重组表达载体和重组病毒构建成功,并在昆虫细胞Sf9中实现了高效表达, 经MTT法测定表达的细胞色素P450nor具有还原NO的生物学活性。     

Degradation of cytochrome P450 2E1: selective loss after labilization of the enzyme.

Mechanism-based inactivation of cytochrome P450 can result in the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. In the present study we took advantage of different modes of inactivation of P450 2E1 by CCl4, 1-aminobenzotriazole, or 3-amino-1,2,4-triazole to investigate parameters which target P450 2E1 for proteolysis from the microsomal membrane. Treatment of mice with CCl4 at the point of maximal induction of P450 2E1 after a single oral dose of acetone resulted in the complete loss of P450 2E1-dependent p-nitrophenol hydroxylation and a 75% loss of immunochemically detectable protein within 1 h of administration. Treatment with 1-aminobenzotriazole at the point of maximal induction caused a complete loss of P450 2E1-dependent p-nitrophenol hydroxylation but only a 12% loss of immunochemically detectable protein 1 h after administration. Treatment of mice with 3-amino-1,2,4-triazole caused a rapid loss of both catalytic activity and microsomal p-nitrophenol hydroxylase activity. However, unlike CCl4 treatment, the activity and enzyme level rebounded 5 and 9 h after treatment. The P450 2E1 ligand, 4-methylpyrazole, administered at the point of maximal induction maintained the acetone-induced catalytic and immunochemical level of P450 2E1. These results suggest that differentially modified forms of P450 2E1 show a characteristic susceptibility to degradation. While there are many potential pathways for protein degradation, the loss of P450 2E1 was associated with increased formation of high molecular weight microsomal ubiquitin conjugates. The formation of ubiquitin-conjugated microsomal protein which correlates with P450 2E1 loss suggests that ubiquitination may represent a proteolytic signal for the rapid and selective proteolysis of certain labilized conformations of P450 2E1 from the endoplasmic reticulum.     

Pregnenolone 16 alpha-carbonitrile-inducible P-450 gene family: gene conversion and differential regulation.

A cytochrome P-450 cDNA clone, designated pP450PCN2, homologous to the previously characterized pregnenolone 16 alpha-carbonitrile (PCN)-induced P-450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P. Hardwick, and C. B. Kasper, J. Biol. Chem. 260:7435-7441), was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P450PCN1 antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P450PCN2 cDNA and protein shared 90% nucleotide and 89% amino acid similarity with P450PCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P450PCN gene. Oligonucleotide probes unique for P450PCN1 and P450PCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P450PCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P450PCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of p450PCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45PCN2 mRNA closely parallel the increases in testosterone 6 beta-hydroxylase activity and P450PCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P450PCN2 mRNA was not significantly induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6 beta-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P450PCN1 and P450PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.