Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.     

Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase,and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver

Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P₁-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P₁-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of ¹⁴C-heme in microsomes prepared from hepatocytes derived from rats given 5¹⁴[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7α-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450_LM4 (molecular weight, 55000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7α-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450_LM4 isolated from phenobarbital- and β-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7α-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450_LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or β-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450_LM4 preparations.     

Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450_e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450_b and P-450_c, have also been highly purified during the isolation of cytochrome P-450_e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450_b, highly purified cytochrome P-450_e is immunochemically identical to cytochrome P-450_b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450_a, P-450_c, P-450_d) or epoxide hydrolase. Purified cytochrome P-450_e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450_b or P-450_d (52,000) but clearly distinct from cytochromes P-450_a (48,000) and P-450_c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450_e is at 450.6 nm, whereas the peak of cytochrome P-450_b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450_e and P-450_b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450_b and P-450_e, respectively. Metyrapone binds to reduced cytochromes P-450_e and P-450_b (absorption maximum at 445–446 nm) but not cytochromes P-450_a, P-450_c, or P-450_d. Metabolism of several substrates catalyzed by cytochrome P-450_e or P-450_b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450_e usually paralleled that of cytochrome P-450_b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450_e was only 15–25% that of cytochrome P-450_b. In contrast, cytochrome P-450_e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450_b. Cytochrome P-450_d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450_a, P-450_b, P-450_c, or P-450_e. The peptide fragments of cytochromes P-450_e and P-450_b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.     

The effects of carbon disulphide on rat liver microsomal mixed-function oxidases, in vivo and in vitro

An intraperitoneal dose of CS₂ (500mg/kg) to male rats resulted in loss of liver microsomal mixed-function-oxidase activity (85% loss of biphenyl 4-hydroxylase), followed by denaturation of liver cytochrome P-450 to cytochrome P-420, and degradative loss of both cytochromes (50% loss). Losses of NADPH–cytochrome c reductase (20%) and cytochrome b₅ were considerably less. Intraperitoneal administration of CS₂ (100mg/kg) to rats pretreated wtih phenobarbitone or 3-methylcholanthrene resulted in similar losses, but the rate of destruction was greater with cytochrome P-450 than with cytochrome P-448. At 12h after intraperitoneal injection of CS₂ to non-pretreated rats, a new cytochrome (P-448) appeared. Rat liver microsomal preparations incubated with CS₂ in the presence of NADPH and O₂ resulted in loss of cytochrome P-450 and mixed-function-oxidase activity directly related to the concentration of CS₂ (10–100μm) and to the period of incubation. Addition of EDTA (1mm) completely inhibited this destruction of cytochrome P-450 by CS₂ in vitro. Addition of CS₂ to liver microsomal preparations resulted in moderate increases in the K_s values for type-I or type-II substrates, but these were insufficient to account for the inhibition of the mixed-function oxidases. We therefore suggest that desulphuration of CS₂ leads to binding of the S to cytochrome P-450, denaturation of cytochrome P-450 to cytochrome P-420, and ultimately to destruction of these cytochromes by autoxidation.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (M_r 120 000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843–850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450_BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450_BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its M_r of 38 000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH₂-terminal amino acid is proline; the penultimate NH₂-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450_BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450_BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450_BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450_BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Fructose utilization and altered cytochrome P-450 in cultured hepatocytes from adult rats

Cultured adult rat hepatocytes incubated in media containing fructose exhibit increased levels of cytochrome P-450, relative to cells incubated with equimolar glucose, and the effect of fructose is proportional to its concentration between 2 and 10 mM. For investigating the mechanism of the effect of fructose on cytochrome P-450 in cultured cells, [U-¹⁴C]fructose or [U-¹⁴C]-glucose were added to the incubation medium, and their uptake and utilization were compared. While the uptake kinetics of the two hexoses were similar, the rate of phosphorylation of fructose was more than 10-fold that of glucose. Similarly, the appearance of fructose carbon in metabolic pools, as well as its conversion to CO₂ and cellular glycerolipid, was increased. The latter finding suggested that fructose might alter cytochrome P-450 by stimulating glycerolipid synthesis, since the stability of the cytochrome is lipid-dependent. However, the changes in glycerolipid formation failed to parallel changes in the level of cytochrome P-450 in fructose-treated cells. Moreover, the relative distribution of ¹⁴C into specific lipids was similar for both hexoses, suggesting that an increased carbon flux in cells incubated with fructose did not directly impose a qualitative change in cellular lipid synthesis. We conclude that the fructose-mediated alteration of cytochrome P-450 in cultured rat hepatocytes reflects a process other than increased incorporation of fructose carbon into metabolic pools.     

Measurement of the metabolism of cytochrome P-450 in cultured hepatocytes by a quantitative and specific immunochemical method

We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [³H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02–0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.     

Avian kidney mitochondrial hemeprotein P-4501α: isolation,characterization and NADPH-ferredoxin reductase-dependent activity

We describe the isolation of cytochrome P-450_1α from chick-kidney mitochondria. Although, gel permeation HPLC yielded 41% of the total amount of P-450 present in cholate-solubilized hemeproteins, it produced a highly purified mixture from which the P-450_1α could be purified to homogeneity in a final detergent-free state by a single-step application of hydrophobic interaction HPLC using hydroxypropyl silica. The purified P-450_1α traveled as a single band in SDS gel electrophoresis with an apparent M_r = 57 000. The absolute spectrum of the P-450_1α(Fe³⁺) form gave a λ_max at 403 nm. This characteristic lends support to the anomalous high-spin heme electron paramagnetic resonance spectrum and the heme structure of P-450_1α which we have previously reported (Ghazarian et al. (1980) J. Biol. Chem. 255, 8275–8281; Pedersen et al. (1976) J. Biol. Chem. 251, 3933–3941). In reconstitution experiments with ferredoxin-dependent NADPH-cytochrome c (P-450) reductase complexes, P-450_1α catalyzed the hydroxylation of 25-hydroxy-9,10-secocholesta-5,7,10(19)-trien-3β-ol at the C-1 position exclusively with a turnover number of 0.03 min^?1. This number is identical to that obtained from measurements of the catalytic activity in intact mitochondria, indicating that only one major species of cytochrome P-450 occurs in chick-kidney mitochondria. The complete responsiveness of cytochrome P-450 concentrations in intact mitochondria to the vitamin D status of chicks provided additional evidence that the major cytochrome P-450 species present in renal mitochondria is uniquely associated with vitamin D metabolism.     

Covalent binding of polychlorinated biphenyls to proteins by reconstituted monooxygenase system containing cytochrome P-450

Incubation in the presence of NADPH and molecular oxygen of ¹⁴C-labeled polychlorinated biphenyls (PCBs) and two tetrachlorobiphenyl (TCB) isomers with a reconstituted system containing NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital(PB)-pretreated rabbits, led to covalent binding of radioactive metabolites of PCBs and TCBs to the protein components of the system. A rabbit liver cytosol fraction added to the system provided more binding sites for the activated metabolites and thus increased the extent of binding markedly. The binding reaction depended absolutely on the reductase, cytochrome P-450 and NADPH, and required dilauroyl phosphatidylcholine and sodium cholate for maximal activity. A further stimulation of the binding was attained by including cytochrome b₅ in the reconstituted system. Four forms of cytochrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCB-binding reaction. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis indicated that, in the system containing the reductase, cytochrome P-450 and cytochrome b₅, PCB metabolites bound to the reductase and cytochrome P-450, but not to cytochrome b₅. In the presence of the liver cytosol fraction, the binding took place to many cytosolic proteins in addition to the reductase and cytochrome P-450.     

Oxidation-reduction potentials of cytochromes P-450 and ferredoxin in the bovine adrenal: Their modification by substrates and inhibitors

The midpoint reduction potentials of the haem iron in bovine adrenal cytochrome P-450 and its associated iron-sulphur protein, adrenal ferredoxin, have been measured, using EPR spectroscopy to monitor the high and low spin ferric haem iron and reduced adrenal ferredoxin signals as a function of potential, in mitochondrial and microsomal suspensions.In mitochondria the high spin (substrate-bound) cytochrome P-450 showed single-component one-electron plots under most conditions; at pH 6.65 cholesterol side-chain cleavage cytochrome P-450 (P-450_scc) had a midpoint E_m = ?305 mV; at pH 8.0 11β-hydroxylase cytochrome P-450 (P-450_11β) had E_m = ?335 mV. Low spin cytochrome P-450 showed more complex titration curves under all conditions, which could be most simply interpreted in terms of two one-electron components with midpoint potentials approx. ?360 and ?470 mV, with varying intensities. During treatments that caused substrate binding, only the ?470 mV component was reduced in magnitude. On sonication and removal of adrenal ferredoxin, the ?470 mV low spin component was converted to higher potential. The potentials could also be altered by the cytochrome P-450 inhibitors aminoglutethimide and metyrapone. In the microsomes, a high spin component of cytochrome P-450 (E_m ≈ ?290 mV) was observed even at pH 8.0, suggesting the binding of an endogenous substrate, while the low spin P-450 showed a predominance of the ?360 mV component. The midpoint potential of membrane-bound adrenal ferredoxin under these various conditions was found to be ?248 mV ± 15 mV.     

Mixed-function oxidase enzymes in adriamycin-sensitive and resistant sublines of P-388 leukemia

Levels of mixed-function oxidase (MFO) enzymes were measured in adriamycin(ADR)-sensitive murine leukemia P-388 and its ADR-resistant subline P-388/ADR. The subcellular fractions of the resistant cells showed decreased contents of MFO components, cytochrome P-450 and cytochrome b₅, in comparison with the identically prepared fractions of the parental tumor. Similarly, the levels of 7-ethoxycoumarin O-deethylase and the rate of ascorbate induced lipid peroxidation in vitro showed lower values in resistant tumor cells than those of P-388 tumor cells. The observed differences in the two tumor cell types were found to be considerably enhanced if the tumor cells were exposed in vitro to ADR before fractionation. The magnitude of induction of the MFO enzymes was significantly greater in the ADR exposed P-388 cells. The corresponding inducibility was suppressed in the drug exposed resistant tumor cells.     

Reconstitution of photosynthetic electron transport and photophosphorylation in cytochrome-c2-deficient membrane preparation of Rhodopseudomonas capsulata.

Cytochrome c₂ was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c₂ could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c₂, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c₂ and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c₂. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O₂. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c₂ is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c₂ from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c₂ was not part of the succinate oxidase system.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats

The effects of retinoic acid (RA) (50 μg/100 g body wt. per day) on hepativ heme oxygenase activty, δ-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T₃ (10 μg/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P < 0.01) and microsomal heme content by 47% (P < 0.001). T₃ administration enhanced heme oxygenase activity by 72% (P < 0.001) and ALAS activity by 251% (P < 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P < 0.001) and heme levels by 75% (P < 0.001). When RA and T₃ were administered together, the retinoid markedly enhanced the T₃ stimulation of heme oxygenase activity; 173% above controls (P < 0.001), and 61% above T₃ alone (P < 0.001). However, RA failed to influence the effect of T₃ on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

Properties of an adrenal cytochrome P-450 (P-450scc) for the side chain cleavage of cholesterol

A highly purified (12 nmol of P-450-heme per milligram of protein) bovine adrenal cortex mitochondrial cytochrome P-450, termed P-450_sce, which cleaves the side chain of cholesterol to yield pregnenolone, is obtained in the substrate-bound ferric form with observed absorption maxima at 393 nm and 645 nm and a shoulder around 540 nm. The absorption spectra of the P-450_scc, whether in the substrate-bound ferric form or in the CO-complexed ferrous form, are subject to environmental perturbation. The addition of adrenal ferredoxin readily restores full ferric high spin type spectrum of the substrate-bound P-450_scc or, together with cholesterol and Tween 20, restores the CO-spectrum of the P-450_scc, exhibiting stable and typical spectra of cytochrome P-450. Tween 20, at concentration of 0.3%, remarkably increases the P-450_scc-catalyzed cholesterol side chain cleavage activity. Based on these findings, a highly reactive and reliable assay has been developed for the conversion of cholesterol to pregnenolone. The specific activity of the P-450_scc, thus determined in the presence of NADPH, NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1), adrenal ferredoxin, cholesterol, and molecular oxygen, is 16 mol of pregnenolone formed per minute per mole of P-450-heme and V of enzyme catalyzed reaction was 30 mol/min/mol of P-450-heme. Apparent K_m values are 120 μm for cholesterol and 1.5 μm for adrenal ferredoxin. The P-450_scc has a pH optimum at pH 7.2 and is most active at ionic strength of 0.1.     

Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal

The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculata mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with g_x values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11β-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spin signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spin parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and high spin ferric g_xy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.     

Ecdysone 20-Monooxygenase Activity of Cytochrome P450 in Ajuga reptans L. Plants and Cell Culture

The concentration of cytochrome P₄₅₀ and ecdysone 20-monooxygenase activity in plants and callus cell culture of carpet bugleweed Ajuga reptans L. were determined. The maximal ecdysone 20-monooxygenase activity of cytochrome P₄₅₀ was found in vegetative rosettes of intact plants. During the stage of flowering, the ecdysone 20-monooxygenase activity of cytochrome P₄₅₀ in plant leaves was higher than in other organs. It was demonstrated that the content of ecdysteroids in callus cell culture is higher than in the intact plant, with concurrent retention of a high ecdysone-20-monooxygenase activity.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Simultaneous purification of a cytochrome P-450 and cytochrome b5 from the house fly,Musca domestica L.

Cytochrome P-450, cytochrome b₅ and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b₅ and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b₅ and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm.Further purification of the anion exchange cytochrome b₅ fraction, by C8 reverse phase HPLC, resulted in a cytochrome b₅ fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa.