Characterization of the protein expressed in Escherichia coli by a recombinant plasmid containing the Bacillus megaterium cytochrome P-450BM-3 gene

Summary In two previous reports (Narhi LO, Fulco AJ, J. Biol. Chem. 261: 7160–7169, 1986; Ibid., 262: 6683–6690, 1987) we described the characterization of a catalytically self-sufficient 119000-dalton P-450 cytochrome that was induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O₂, this polypeptide (cytochrome P-450_BM-3) catalyzed the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase was cloned into Escherichia coli and the clone harboring the recombinant plasmid produced a protein that behaved electrophoretically and immunochemically like the B. megaterium enzyme (Wen LP, Fulco AJ, J. Biol. Chem. 262: 6676–6682, 1987). We have now compared authentic P-450_BM-3 from B. megaterium and putative P-450_BM-3 isolated from transformed E. coli and have found them to be indistinguishable with respect to chromatographic and electrophoretic behavior, reaction with specific antibody, prosthetic group (heme, FAD and FMN) analyses, spectra, enzymology, limited trypsin proteolysis and partial amino acid sequencing. We thus conclude that the P-450 cytochrome expressed by the transformed E. coli is essentially identical to native P-450_BM-3 induced by barbiturates in B. megaterium. The evidence furthermore suggests that the primary amino acid sequence of this complex protein is alone sufficient to direct the proper integration of the three prosthetic groups and to specify folding of the polypeptide into the correct tertiary structure.Abbreviations SDS Sodium Dodecylsulfate - PAGE Polyacrylamide Gel Electrophoresis - HPLC High Performance Liquid Chromatography     

Ecdysone 20-monooxygenase in a cricket,Gryllus bimaculatus (Ensifera,Gryllidae)—characterization of the microsomal midgut steroid hydroxylase in adult females

Summary Ecdysone 20-monooxygenase, the enzyme system which converts ecdysone into 20-hydroxyecdysone, was characterized in the midgut of 4-day-old female adult Gryllus bimaculatus using an in vitro radioassay. Differential centrifugation and sucrose gradient centrifugation revealed that ecdysone 20-monooxygenase activity is associated with the microsomal fractions. The 20-monooxygenase was found to be most active in potassium phosphate buffer, pH 7.8, at an osmolarity of 100 mOsm and at 39 °C assay temperature. The conversion of ecdysone into 20-hydroxyecdysone was linear over an incubation period of 12 min and with respect to a protein concentration of 3 mg·ml^–1. K⁺ and Na⁺ (10^–3–10^–1 M), Ca²⁺ (2.3 mM), and EDTA (1–5 mM) did not affect monooxygenase activity, whereas Mg²⁺ (2.3–10 mM) slightly inhibited enzyme activity. The enzyme complex has an apparent K_m for ecdysone of 3.7·10^–7 M and is competitively inhibited by its product, 20-hydroxyecdysone, with an apparent K_i of 4·10^–6 M. The cytochrome P-450 nature of the steroid hydroxylase was shown by its obligate requirement for NADPH and its inhibition by carbon monoxide, metyrapone, and p-chloromercuribenzoate, but not by cyanide. The insect systemic growth disruptor, azadirachtin, was found to inhibit ecdysone 20-monooxygenase activity with a I₅₀ of 8·10^–4 M. From the CO-difference spectrum, a cytochrome P-450 content of 285 pmol·mg protein^–1 was calculated for midgut microsomes of 4-day-old females.Abbreviations GO carbon monoxide - EDTA ethylenediamine tetraacetic acid - HPLC high performance liquid chromatography - I ₅₀ concentration for 50% inhibition - KCN potassium cyanide - K ₁ inhibition constant - K _m Michaelis-Menten constant - MOPS 3-morpholinopropanesulfonic acid - NADH/NAD ⁺ nicotinamide adenine dinucleotide reduced/oxidized - NADPH/NADP ⁺ nicotinamide adenine dinucleotide phosphate reduced/oxidized - Na ₂ S ₂ O ₄ sodium dithionite - SEM Standard error of mean - TLC thin-layer chromatography - TRIS 2-amino 2-hydroxymethyl-1,3-propanediol (trishydroxymethyl aminomethane) - V _max maximal reaction velocity     

Influence of the growth substrate and the oxygen concentration in the medium on the cytochrome P-450 content in Candida guilliermondii

Summary The induction of cytochrome P-450 in Candida guilliermondii by different growth substrates was investigated. Hexadecane is a strong inducer, whereas its oxidation products induce only weakly. It could be shown that the oxygen concentration in the medium influences the cytochrome P-450 content in cells growing on hexadecane. At oxygen concentrations between 100% and 15% of saturation the cytochrome P-450 content reaches 25–35 nmol/g dry weight. Reducing the oxygen to lower concentrations increase the cytochrome P-450 content up to approximately 100 nmol/g dry weight.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including microsomes, described above, and mitochondria. The amount of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40–50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20–30 sec of incubation with deoxycholate.The content of RNA, phospholipids, and cytochromeb ₅ in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.     

Microsomal cytochromes of Candida tropicalis grown on alkanes

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

Degradation of tributyltin by the filamentous fungus Cunninghamella elegans, with involvement of cytochrome P-450

Cunninghamella elegans degraded tributyltin (TBT) at 20 mg l^–1 when grown in Sabouraud medium. Above this concentration, growth was inhibited. After 7 d 70% TBT (added at 10 mg l^–1) was converted to less toxic derivatives: dibutyltin and monobutyltin. TBT metabolism was totally blocked by cytochrome P-450 inhibitors, metyrapone and proadifen. Only in medium with 1-aminobenzotriazole, was dibutyltin (0.42 mg l^–1) found after 7 d of culturing. It is postulated that the significant resistance of C. elegans to TBT is associated with the capacity of the fungus to metabolise TBT.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

The carbon monoxide- and oxygen-reacting haemoproteins of Streptomyces clavuligerus: cytochrome aa 3 is the predominant terminal oxidase of the respiratory chain

The nature of the carbon monoxide- and oxygen-reacting haemoproteins in the respiratory chain of the filamentous antibiotic-producing bacterium Streptomyces clavuligerus has been investigated. CO-difference (i.e. CO+ reduced minus reduced) spectra of intact cells showed the presence of cytochrome aa ₃, a CO binding b-type cytochrome, and a pigment resembling cytochrome d. In addition, cells that were approaching the end of the growth phase showed the presence of cytochrome P450: this pigment was undetectable in cells harvested early in the growth cycle. High speed centrifugation of cell-free extracts prepared from cells broken by sonication showed that cytochrome aa ₃ was tightly membrane-bound and that cytochrome P450 was soluble. Inhibition of oxygen uptake rates of cells by cyanide indicated that one component, which showed 50% inhibition at 2–4 mM CN^–, was acting as major terminal oxidase: this was observed in cells harvested from all stages of growth. Photodissociation (i. e. photolysed, CO reduced minus CO reduced) spectra at-118°C, in the absence of oxygen, showed cytochrome aa ₃ to be the sole photolysable CO-reacting haemoprotein. At higher temperature (-87°C), in the presence of oxygen, cytochrome aa ₃ formed a complex with oxygen that could not be photolysed by similar intensities of light. By raising the temperature to-43°C, the oxidation of c-type cytochromes was observed. It is concluded that cytochrome aa ₃ is the predominant terminal oxidase in S. clavuligerus and that the other CO reacting haemoproteins, of unknown function, are unlikely to be oxidases.     

Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles

When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450_LM2, cytochrome b₅ enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or $\text{p}$-nitroanisole about 5-fold. Cytochrome b₅ did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b₅-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H₂O₂ or $\text{O}{}_2{}^{\mathrm{?}}$ in the system, thus strongly indicating cytochrome b₅ being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase.     

Effect of calcium(ion) uptake by rat adrenal mitochondrial on pregnenolone formation and spectral properties of cytochrome P-450

The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenalgglands. In the presence of succinate, addition of calcium chloride in the concentration range 20–150 μM caused an inhibition of pregnenolone formation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP⁺. Under these conditions, calcium chloride in the concentration range 120–875 μM caused a 2–4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone.The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium the interaction of mitochondrial cytochrome P-450 with endogenous substrate.     

Slaving the cytochrome P-450 dependent monooxygenase system by periodically applied light pulses

The light-induced enhancement of 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system consisting of the enzyme P-450 II B1 (P-450_PB-B) and the NADPH-cytochrome P-450 reductase. The phases of the catalytic cycle of 2 · 10¹² protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.2–2.5 s repetition time, and 390–470 nm 0.27 Joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes worked in phase if the repetition time (1.32 s) was slightly smaller than the catalytic cycle time of the free running enzyme (1.54 s). The percentage of synchronized enzyme complexes as a function of the repetition time is shown. It is shown that the lifetime of the product-enzyme complex is shortened by the light.Abbreviations P-450 liver microsomal cytochrome P-450 - PB phenobarbital Offprint requests to: H. Gruler     

The responses of hepatic monooxygenases of guinea pig to cadmium and nickel

When Cd (3.58 mg CdCl₂·H₂O/kg, ip) was administered to male guinea pigs 72 h prior to sacrifice, the metal significantly inhibited the aniline 4-hydroxylase (AH) (16%), ethylmorphoneN-demethylase (EMND) (26%), and aminopyrineN-demethylase (AMND) (18%) activities and cytochrome P-450 (12%) and cytochrome b₅ (10%) levels. Cd did not alter the hepatic microsomal heme level. Cd, however, significantly increased the hepatic microsomalp-nitroanisoleO-demethylase (p-NAOD) (53%) activity. When Ni (59.5 mg NiCl₂·6H₂O/kg, sc) was administered to the guinea pigs 16 h prior to sacrifice, the metal significantly depressed AH (49%),p-NAOD (66%), EMND (47%), and AMND (37%) activities, and cytochrome P-450 (15%), cytochrome b₅ (24%), and microsomal heme (28%) levels. For the combined treatment, animals received the single dose of Ni 56 h after the single dose of Cd and then were killed 16 h later. In these animals, significant inhibitions were noted in AH (51%), EMND (47%), and AMND (30%) activities, and cytochrome P-450 (15%), cytochrome b₅ (26%), and microsomal heme (30%) compared to those of controls. In the case ofp-NAOD activity, the influence was in favor of Ni, i.e, the inhibition was about 61% by the combined treatment. These results reveal that:

The plant growth regulator activity of the fungicide,epoxiconazole, on Galium aparine L. (cleavers)

The plant growth regulator activity of epoxiconazole, a new triazole fungicide, was investigated by time-course, dose-response and histology experiments with Galium aparine L. (cleavers). Seven days after treatment with 125g ai ha^–1 epoxiconazole (field rate), plant height was reduced by 43%. After seventeen days, leaflet area was reduced by 27% but leaflet fresh weight was not significantly influenced. This was partly because leaflet thickness had increased by 20% following epoxiconazole application. Chlorophyll concentrations were also increased on a unit area basis. Examination of leaflet anatomy showed that epoxiconazole elongated palisade, spongy mesophyll and upper epidermal cells. For example, 125g ai ha^–1 caused a 35% increase in the length of spongy mesophyll cells. Epoxiconazole also prevented cell separation as there were significantly more palisade and spongy mesophyll cells per unit area than in leaflets sprayed with water. Stem development was reduced and 125g ai ha^–1 inhibited the elongation of pith cells in stem tissue by 53%. However, the simultaneous application of gibberellin A₃ (GA₃) with epoxiconazole resulted in stem elongation similar to that of control plants. These observations are consistent with the expected effects following the inhibition of cytochrome P-450 dependent enzyme activity.Abbreviations GA₃ gibberellin A₃ - g ai ha^–1 grams of active ingredient per hectare - L ha^–1 litres per hectare - PPFD photosynthetic photon flux density - RH relative humidity - SE standard error     

Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine ($\text{o}$-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-$\text{p}$-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-$\text{p}$-aminoglutethimide exhibited an I₅₀ value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.     

Inhibition of the benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene-induced toxicity by allyl sulfides in human epidermal keratinocytes

Diallyl sulfide (DAS) and diallyl disulfide (DADS) at 25 g ml^–1decreased the benzo[a]pyrene (B[a]P)-induced colony growth inhibition of human epidermal keratinocytes. DAS and DADS decreased B[a]P-DNA and B[a]P-protein adducts by 65% and 49–55%, respectively. The B[a]P-induced ethoxyresorufin O-deethylase activity, a marker enzyme for cytochrome P450 1, was decreased from 3 to 1.7–1.9 nmol min^–1 mg^–1 microsomal protein by DAS and DADS treatments. The activity of glutathione S-transferase, a detoxifying enzyme for B[a]P, but was decreased by DADS, but was unaffected by DAS.     

Microsomal flavonoid 3'-monooxygenase from maize seedlings

Identification of flavonoid 3′-monooxygenase establishes another reaction in the biosynthesis of flavonoid compounds in maize (Zea mays L.). The flavonoid 3′-hydroxylase was obtained as a microsomal enzyme preparation by buffer extraction of 5 day old maize seedlings and ultracentrifugation. Seedlings were exposed to light 24 hours prior to enzyme extraction. The extraction buffer required the addition of sucrose or glycerin and dithiothreitol to obtain an active hydroxylase that retained its activity on storage at −70°C. Enzymic activity required O₂ and NADPH, was optimum at pH 8.5 and 30°C, and could be inhibited 79% by carbon monoxide. Carbon monoxide inhibition could be reduced to 21% by irradiation of the samples with 450 nanometer light during incubation. Kaempferol, a flavonol; naringenin, a flavanone; and apigenin, a flavone, all served as substrates for the hydroxylase. Treatment of the microsomal enzyme preparation, previously reduced with sodium dithionite, with carbon monoxide gave a 455 nanometer absorption peak which disappeared on oxidation of the preparation with the formation of a 420 nanometer peak. These results suggest a cytochrome P-450 type monooxygenase enzyme. The concentration of cytochrome P-450 was 0.21 nanomoles per milligram protein. Identification of the monooxygenase provides further biochemical information about a biosynthetic sequence for which the genetics have been studied intensely.     

Cytochrome P-450 and alkane hydroxylase activity in Candida guilliermondii.

In the investigated Candida guilliermondii strain after growth on n-alkanes as the only carbon and energy source 5--10 nMol cytochrome P-450 per g cells (wet weight) could be detected. Cytochrome P-450 and alkane hydroxylase activity was found in the 100 000 xg pellet. Cofactor studies and inhibition experiments revealed the existence of a NADPH-dependent cytochrome P-450 alkane hydroxylase system.     

Involvement of cytochrome P-450 in the 15α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by Penicillium raistrickii

The 15α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione (GD) with different subcellular fractions of Penicillium raistrickii i 477 was investigated. Cytochrome P-450 was shown to be involved in this reaction. The steroid transformation was inhibited by carbon monoxide, metyrapone, p-CMB, iodoacetamide, N-methylmaleimide and several metal ions. The 15α-hydroxylase was observed to be dependent on nicotinamide-adenine dinucleotide phosphate (NADPH) replaceable by NaIO₄, and the activity was enhanced by a NADPH-regenerating system, indicating the involvement of the NADPH-cytochrome c (P-450) reductase. This was further confirmed by the inhibition of the hydroxylase activity in the presence of cytochrome c. No effect was observed in the presence of azide and antimycin A. Solubilized microsomes gave an absorption maximum at 453 nm in carbon monoxide difference spectrum, and showed a Type-I GD-binding spectrum typically for cytochrome P-450 interaction with substrate. First results about the inducibility of the enzymes involved in the 15α-hydroxylation of GD are shown.     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

Orientation of reaction center complexes fromRhodobacter sphaeroides in proteoliposomes and the effect ofo-phenanthroline on electrogenesis during primary photochemical reaction

The orientation ofRhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown thato-phenanthroline, an inhibitor of electron transfer between primary (Q_A) and secondary (Q_B) quinone acceptors, can also inhibit the photoinduced Q_A reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-inducedo-phenanthroline inhibition of primary dipole (P-870⁺ · Q _A ^– ) formation is of a competitive nature.