Enzyme activity of the cytochrome P-450 monooxygenase system in the presence of single chain lipid molecules.

The influence of single chain lipids on the 7-ethoxycoumarin O-deethyase activity of the reconstituted binary protein complex of isolated cytochrome P450 and NADPH-cytochrome P450 reductase has been examined. The enzyme activity of this binary enzyme complex has been shown to be influenced by (i) altering the complexation process of both proteins, (ii) by altering the catalytic cycle time of the active binary protein complex and (iii) by altering the fraction of substrate molecules at the catalytic center of the enzyme. Competitive inhibition was measured for all single chain molecules. The following dissociation coefficients of substrate and lipids used for the catalytic center of the protein were obtained: 110 microM 7-ethoxycoumarin (substrate), 1.1 microM MOG (1-monooleoyl-rac-glycerol), 0.3 microM SPH (D-sphingosine), 1.5 microM OA (oleic acid), 3.0 microM LPC (L-alpha-lysophosphatidyl-choline), 15.5 microM MSG (1-monostearoyl-rac-glycerol), 9.5 microM AA (arachidonic acid), 9.0 microM PaCar (palmitoyl-L-carnitine), 3.5 microM MPG (2-monopalmitoyl-glycerol), 1.5 microM LPI (L-alpha-lysophosphatidyl-inositol), 50 microM LA (lauric acid), 60 microM MA (myristic acid), 85 microM PA (palmitic acid), >100 microM SA (stearic acid). Only competitive inhibition with the substrate molecule 7-ethoxycoumarin was observed for the single chain lipids LA, MA, PA, SPH, SA, and OA. Non-competitive effects were observed for MPG (-0.03 microM(-1)), PaCar (-0.02 microM(-1)), MSG (-0.023 microM(-1)), LPC (-0.03 microM(-1)), AA (-0.03 microM(-1)), and MOG (+0.04 microM(-1)). The negative sign indicates that the cycle time of the working binary complex is enlarged. The positive sign indicates that the formation of the binary complex is enhanced by MOG.

Complexation of membrane-bound enzyme systems.

The effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time tau =2.65 +/- 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+[reductase] <--> [complex]) described by the dissociation constant KD, at high substrate concentration ([S]>>KS) and low product concentration ([P]<

Investigations on the role of cytochrome b5 and divalent cations in the maximal nifedipine oxidase activity of human liver.

Native human cytochrome P4503A4 was most active in nifedipine oxidation when incorporated into a binary phospholipid vesicular system with human NADPH-cytochrome P450 reductase. The turnover numbers were estimated to be 17.6 and 19.6 min-1 in the presence of Mg2+ or Ca2+ ions (5 mmol/l) in the test system, respectively. Inclusion of b5 in the vesicular CYP3A4: reductase system results in a slightly lower nifedipine oxidase activity of 16.9 min-1 in the presence of Mg2+ ions. These results demonstrate that b5 is not an essential component in CYP3A4 catalyzed nifedipine oxidation in human liver.

Blue light mediated photoreduction of the flavoprotein NADPH-cytochrome P450 reductase. A Förster-type energy transfer.

The absorption spectra and the corresponding molar absorption coefficients of the fluorophores umbelliferone, FAD and FMN and of the FAD and FMN containing flavoprotein NADPH-cytochrome P450 reductase of different oxydation-reduction states are documented. Binding spectra of the ligand umbelliferone with the CYP2B1:NADPH-cytochrome P450 reductase-complex were determined by difference spectroscopy. The Scatchard plot of the equilibrium ligand binding shows a high affinity part and a low affinity part of 12 and 34 umbelliferone binding sites per CYP2B1:reductase-complex molecule, respectively. The fluorescence excitation and emission spectra of the donor molecule umbelliferone and the acceptor molecules FAD and FMN are given. The fluorescence spectra of the reaction components under test conditions of CYP2B1-dependent 7-ethoxycoumarin-O-deethylase are measured. The excitation energy transfer from the donor umbelliferone (lambda E = 380 nm; lambda F = 460 nm) to the acceptor molecule FMN (lambda E = 465 nm; lambda F = 525 nm) was examined under assay conditions. The results demonstrate that a radiationless Förster-type energy transfer takes place in the presence of the CYP2B1:reductase-complex. It turned out that this effect is a function of the protein complex-concentration. The data presented here combined with previously made observations by Müller-Enoch (Müller-Enoch D. (1994), Z. Naturforsch. 49c, 763-771) support the finding that the umbelliferone molecules, n = 12-34, bound per mole of CYP2B1:reductase-complex, transfer their absorbed light energy radiationless to the FAD binding domain. The complex formed containing 12 or 34 molecules of umbelliferone provides absorption coefficient values at lambda = 380 nm of 78 and 221 mM-1.cm-1, respectively. The Förster-type energy transfer from the donor umbelliferone to the acceptor FAD not only leads to a light activation of the singlet state of FAD but also to a conformational change of the amino acids close to the FAD binding side to favour the encaging of the FAD* triplet state which reacts with the NADPH to form the FADH2 reductase. Due to this process the overall reaction can start with the unquenched excited FAD* triplet state as an intermediate which is about 30 kJ/mol lower in energy than the dark reaction.

Product autofluorescence activation in the cytochrome P450 monooxygenase system.

The relative increase of the 7-ethoxycoumarin-O-deethylase and the 6,7-dimethoxycoumarin-O-demethylase activities were found 93 and 236% using a reconstituted cytochrome P4502B1:NADPH-P450 reductase system by adding to the reaction mixtures their own products. The assays were irradiated during the reactions with the excitation wavelength maximum of their products umbelliferone (lambda E = 365 nm) or scopoletin (lambda E 98 nm), respectively. Addition of the products to the reaction mixtures without irradiation (dark reaction) had no activating effect on the specific activities of the 7-ethoxycoumarin-O-deethylase or the 6,7-dimethoxycoumarin-O-demethylase. The relative increase of the specific activities is dependent on the excitation light intensities and was at maximum when the light intensity at the sample cuvette was 0.4 mW/cm2. The activation energies of the P4502B1-dependent 7-ethoxycoumarin-O-deethylation reaction obtained from Arrhenius plots with and without added umbelliferone and irradiation with lambda E = 365 nm are 14.7 kJ/mol and 33.5 kJ/mol, respectively, in the temperature range of 27-37 degrees C. The irradiation energy of the fluorescent product umbelliferone change the catalytic mechanism, which has a two times lower activation energy in the presence of the irradiated product umbelliferone. Umbelliferone and scopoletin have highest fluorescence intensities in the wavelength range of the blue light (440-480 nm). The photochemical action spectrum of the 7-ethoxycoumarin-O-deethylase of the P4502B1:reductase system is also found to be in the wavelength range of 420-470 nm. No activation effect was seen with irradiating light lower than 400 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse liver cytochrome P-450 (P-450IIIAM1): its cDNA cloning and inducibility by dexamethasone.

A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone.

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-450PB-B) and the NADPH-cytochrome P-450 reductase. The phases of the catalytic cycle of 2 x 10(12) 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.

Light-induced activation and synchronization of the cytochrome P-450 dependent monooxygenase system.

The light-induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in a reconstituted system, consisting of the enzyme P-450PB-B and the NADPH-cytochrome P-450 reductase. The relative increase of the activity was about 15%. It is shown that the product release process is accelerated by light. The phases of the catalytic cycle of 2 x 10(12) protein complexes were locked by periodic application of light pulses (0.1 s duration, 1.32 s repetition time, and 390-470 nm, 0.27 joule/nmol P-450). More than 80% of the active reconstituted enzyme complexes (= "molecular machines") worked in phase after a few light pulses. The phase relation continued even after switching off the light pulses. The catalytic cycle time was 1.54 s, giving a turnover number of 39 min-1. The turnover number, as determined from the enzyme activity under optimum conditions, was 39 min-1. Due to the dissociation constant of the P-450PB-B:NADPH-P-450 reductase complexes [3] only 24% of the proteins were in the active (= working) state under the conditions used. The lifetime of this complexes is larger than 6 s since more than 4 cycles of the free running enzyme can be observed. This is the first report, that all catalytic active complexes in the test tube can be synchronized by an external light source, if the right repetition time of the pulses is chosen, so that all these "molecular machines" work in phase.

Differentiation of cytochrome P-450 activities with scoparone as substrate. A rapid one-step HPLC-analysis.

A direct and highly sensitive high performance liquid chromatography (HPLC) method for measuring cytochrome P-450 activities in biological probes, with the substrate scoparone, is described. Using 10-100 microliter of a supernatant of an incubation mixture of scoparone (0.1 mmol/l), microsomal fractions (1 nmol P-450/ml), MgCl2 (2 mmol/l), and NADPH (4 mmol/l), the products scopoletin and isoscopoletin as well as the substrate scoparone were separated by HPLC on an ODS-Hypersil RP-18 column. The compounds were detected by a UV-spectrophotometer (345 nm) and quantified by the aid of an external calibration curve. The limits of detection were 10 pmol for the products scopoletin and isoscopoletin. This method allows the direct quantification of P-450 activities and the simultaneous differentiation between "3-methylcholanthren-like" or "phenobarbital-like" induction states by estimation of the scopoletin: isoscopoletin ratios.

Oxidation of quinidine by human liver cytochrome P-450.

The anti-arrhythmic quinidine has been reported to be a competitive inhibitor of the catalytic activities of human liver P-450DB, including sparteine delta 2-oxidation and bufuralol 1'-hydroxylation, and we confirmed the observation that submicromolar concentrations are strongly inhibitory. Human liver microsomes oxidize quinidine to the 3-hydroxy (Km 4 microM) and N-oxide (Km 33 microM) products, consonant with in vivo observations. Both bufuralol and sparteine inhibited microsomal quinidine 3-hydroxylation. Liver microsomes prepared from DA strain rats showed a relative deficiency in quinidine 3-hydroxylase activity in females compared to males. These observations might suggest that quinidine oxidation is catalyzed by the same P-450 forms that oxidize debrisoquine, bufuralol, and sparteine; i.e., rat P-450UT-H and P-450DB. However, neither of these two purified enzymes catalyzed quinidine 3-hydroxylation, and anti-P-450UT-H, which strongly inhibits human liver microsomal bufuralol 1'-hydroxylation, did not substantially inhibit quinidine 3-hydroxylation or N-oxygenation. P-450MP, the human S-mephenytoin 4-hydroxylase, also does not appear to oxidize quinidine but P-450NF, the human nifedipine oxidase, does. Anti-P-450NF inhibited greater than 95% of the 3-hydroxylation and greater than 85% of the N-oxygenation of quinidine in several microsomal samples. Quinidine inhibited microsomal nifedipine oxidation and, in a series of human liver samples, rates of nifedipine oxidation were correlated with rates of quinidine oxidation. Thus, quinidine oxidation appears to be catalyzed primarily by P-450NF and not by P-450DB. Quinidine binds 2 orders of magnitude more tightly to P-450DB, which does not oxidize it, than to P-450NF, the major enzyme involved in its oxidation. The substrate specificity of human P-450NF is discussed further in terms of its regioselective oxidations of complex molecules including quinidine, aldrin, benzphetamine, cortisol, testosterone and androstenedione, estradiol, and several 2,6-dimethyl-1,4-dihydropyridines.

The activation of the cytochrome P-450 dependent monooxygenase system by light.

The cytochrome P-450 dependent monooxygenase system of rat liver microsomes is investigated by light dosimetry (action spectroscopy). The scoparone-O-demethylation activity is enhanced by light and depends on the wavelength of the irradiating light. The relative increase of the activity (about 15%) by the irradiating light (approximately 0.5 mW/cm2) is maximal at a wavelength of 400 nm. The light induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in reconstituted systems, consisting of one of two P-450 enzymes (P-450 beta NF-B and P-450 PB-B) with the NADPH-cytochrome P-450 reductase. The action spectrum of the reconstituted P-450 beta NF-B:NADPH-P-450 reductase complex shows a maximum between 420 and 440 nm. The relative increase of the activity induced by light of 420 nm was 7.3% and 9% for the reconstituted systems of P-450 beta NF-B and P-450 PB-B, respectively. The results are discussed in analogy to the classic experiments of Warburg with its blocked CO-enzyme-complex. The results can best be explained by the assumption that the light induced enhancement of the enzyme activity is due to an excitement of those intermediate states of the P-450 catalytic cycle (ferric and ferrous state of the heme iron) which are rate limiting.

Regioselective O-demethylation of scoparone: differentiation between rat liver cytochrome P-450 isozymes.

The ratios of the scoparone O-demethylation products scopoletin to isoscopoletin were determined for reconstituted complexes of NADPH-P-450 reductase and each of four P-450 isozymes in a 2:1 molar ratio with a 1:1 mixture of [7-O-methyl-14C]- and [6-O-methyl-14C]-scoparone as substrate. The two phenobarbital inducible forms P-450PB-B and P-450PB-D have a 1:0.8 +/- 0.05 scopoletin to isoscopoletin ratio, and the two beta-naphthoflavone inducible forms P-450 beta NF-B and P-450 beta NF/ISF-G have ratios of 1:4.4 +/- 0.1 and 1:3.8 +/- 0.1, respectively. The scoparone-O-demethylation activities of the reconstituted preformed complexes of the four P-450 isozymes are given.

Induction of cytochrome P-450 isozymes in liver microsomes of rats treated with the cardiotonic agent sulmazole.

Pretreatment of adult male rats with sulmazole (AR-L 115 BS) results in a 2-6-fold increase of the liver microsomal scoparone O-demethylation and 7-ethoxycoumarin O-deethylation activity. The change in the ratio of the demethylation products scopoletin to isoscopoletin from 1 : 1.8 +/- 0.1 in control microsomes to 1: 2.5 +/- 0.1 in microsomes from sulmazole pretreated rats is statistically significant. Sulmazole produces a modified type II difference spectrum when added to microsomes of control or sulmazole-pretreated rats. Immunoquantitation of seven cytochromes P-450 showed that two forms, namely P-450 beta NF/ISF-G and P-450 beta NF-B, are increased 3-4-fold in the microsomes of sulmazole-pretreated rats.

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid.

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.

O-demethylation of scoparone and studies on the scoparone-induced spectral change of cytochrome P-450 in rat liver microsomes.

Scoparone (6,7-dimethoxycoumarin) is demethylated to scopoletin (7-hydroxy-6-methoxycoumarin) and isoscopoletin (6-hydroxy-7-methoxycoumarin) by the cytochrome P-450-dependent monooxygenase system of rat liver microsomes. Under the conditions used, the ratio of scopoletin to isoscopoletin was determined to 1:1.8 +/- 0.1 for microsomes from untreated rats. Based on this reaction, a direct fluorometric method for the microsomal O-demethylation activity for scoparone is described. The fluorescence of the scopoletin formed in the incubation mixture is recorded after the adjustment of the excitation and emission wavelengths to 398 and 460 nm, respectively. The fluorescence of scoparone and isoscopoletin does not interfere with the test. Pretreatment of rats with phenobarbital or polycyclic hydrocarbons (3-metylcholanthrene, benzo[a]pyrene) causes a change in the ratio of the demethylation products scopoletin to isoscopoletin which was determined to be 1:2.5 +/- 0.1 (benzo[a]pyrene or 3-methylccholanthrene) or 1:5.9 +/- 0.01 (phenobarbital) respectively, and a significant increase in the amount of microsomal O-demethylation activity. Thus the ratio of the two products varies significantly with the state of induction. The difference spectra of scoparone with liver microsomes obtained from benzo[a]pyrene- and 3-methylcholanthrene-pretreated rats show an absorption peak at 416 nm and a trough at 393 nm with an isosbestic point at 405 nm. This scoparone-induced modified Type II spectral change seems to indicate the interaction of the 6- or 7-methoxy group of scoparone with the heme ion of the 394-nm form and its conversion into a modified ferrihemochrome with a absorption peak at 416 nm. This is modified ferrihemochrome is not identical with the 418-nm form of the cytochrome P-450.

[A fluorometic test for microsomal monooxygenase activity in the rat liver with scoparone as substrate (author's transl)]. / Eine fluorometrische Bestimmungsmethode für mikrosomale Monooxygenase-Aktivität der Rattenleber mit Scoparon als Substrat.

A sensitive fluorometric test for the determination of monooxygenase activity in liver microsomes from rats is described. The assay is based on the O-demethylation of 6,7-dimethoxycoumarin (scoparone) to 7-hydroxy-6-methoxycoumarin and 6-hydroxy-7-methoxycoumarin. Pretreatment of rats with phenobarbital or polycyclic hydrocarbons (benzo[a]pyrene, 3-methylcholanthrene) causes a significant increase in the amount of microsomal dealkylation activity.

[A sensitive fluorometric determination of catechol methyltransferase activity (author's transl)]. / Eine empfindliche fluorometrische Bestimmungsmethode für die Catechol-Methyltransferase

A method for the determination of catechol-methyltransferase activity is described, based on the measurement of fluorometric intensity of 7-hydroxy-6-methoxycoumarin (scopoletin), enzymatically produced by dihydroxycoumarin in the presence of the methyl donor S-adenosylmethionine.

[O-Methylation of epinephrine, 3,4-dihydroxybenzoic acid and 6,7-dihydroxycoumarin in yeasts (author's transl)]. / O-Methylierung von Adrenalin, 3.4-Dihydroxybenzoesäure und 6.7-Dihydroxycumarin in Sprosspilzen

In the yeasts C. albicans, C. tropicalis, and C. stellatoidea but not in C. krusei, R.rubra, and S. cerevisiae enzyme activity was found by which--as by the catechol-O-methyltransferase (EC 2.1.1.6) found in the liver--the O-methylation of epinephrine to metanephrine and paranephrine, of 3,4-dihydroxybenzoic acid to 4-hydroxy-3-methoxybenzoic acid and 3-hydroxy-4-methoxybenzoic acid, and of 6,7-dihydroxycoumarin to 7-hydroxy-6-methoxycoumarin and 6-hydroxy-7-methoxycoumarin is catalysed. When the substrates 3,4-dihydroxybenzoic acid, or 6,7-dihydroxycoumarin or epinephrine were incubated in the presence of S-adenosyl-L-[methyl-14C]methionine and S-adenosylmethionine hydrogensulfate with a 100 000 X g supernatant of C. albicans, C. tropicalis or C. stellatoidea the corresponding O-methylethers were detected in the extracts of the incubation medium by thin-layer chromatography. Final identification of the isomeric radioactive O-methylethers obtained from 3,4-dihydroxybenzoic acid and 6,7-dihydroxycoumarin was performed after thin-layer chromatographic separation by the reversed isotope dilution technique. The radioactive m- and p-O-methyl derivatives from epinephrine were separated by thin-layer chromatography and then cleaved with periodate to the corresponding aldehydes which were also identified mainly by the reversed isotope dilution technique.

[6.7-Dihydroxycoumarin (Aesculetin) as a substrate for catechol-o-methyltransferase (author's transl)]. / 6.7-Dihydroxycumarin (Aesculetin) als Substrat der Catechol-O-Methyltransferase

6,7-Dihydroxycoumarin (Aesculetin) was found to be a substrate of rat liver Catechol-O-methylfransferase (COMT) (EC 2.1.1.6). Incubation of this substrate with S-Adenosyl-L-[methyl-14C]methionine and/or S-Adenosylmethionin-hydrogensulfate in the presence of COMT yields the highly fluorescent compounds 7-hydroxy-6-methoxycoumarin (Scopoletin) and 6-hydroxy-7-methoxycoumarin (Isoscopoletin) in the ration of about 2:1. The O-methylated products obtained from Aesculetin were identified after separation by thin layer chromatography mainly by the reversed isotope dilution technique. The fluorescence of the isolated methylethers was proportional to concentration within the range from 10(-5)-10(-7) M. A reciprocal plot of activity versus substrate concentration gives a Km of 1 X 10(-6) M.