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1Author    Dieter Müller-Enoch3, Hans GrulerbRequires cookie*
 Title    Complexation of Membrane-Bound Enzyme Systems  
 Abstract    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 x =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 K D at high substrate concentration ([5]»/C5) and low product con­ centration ([ P ] « /^) is determined to be /CD/[P450]o = 0.3 and 2.0, respectively. These values reflect large differences in the affinity of both P450 forms for the same type of reductase which is only due to their modified membrane-binding domains. In the present work, it has been shown for the first time, that the membrane-binding domain of cytochrome P450 en­ zymes determines the complexation process of the binary P450:reductase system. Further­ more, the nifedipine oxidase activity of the human CYP3A4 form reconstituted with two different types of reductase from human and rabbit also has the same catalytic cycle time x = 2.65 ± 0.15 s. This result is based on the similarity of the primary structure of the cytosolic catalytic domain of both reductase types. However, the complex was formed with different dissociation constants of A T D/[P450]o = 0.3 and 4.7, respectively. This different affinity of both reductase types to the same P450 form is interpreted as a consequence of the substantial alteration of the amino acids in the N-terminal primary structure of their membrane-binding domains. 7-Ethoxycoumarin O-deethylase activity of two rat P450 forms (CYP2B1 and CYP1A1) were reconstituted with the same rat reductase. The catalytic cycle time for each P450 form is x = 1.8 and 0.6 s, respectively. Correspondingly, the complex formation process controlled by the dissociation constant K n has changed from /CD/[P450]o = 2.3 to 1.7, respec­ tively. This is because both forms differ in their cytosolic as well as in their membrane-binding domains. 
  Reference    Z. Naturforsch. 55c, 747—752 (2000); received April 13/May 20 2000 
  Published    2000 
  Keywords    Cytochrome P450, NADPH-Cytochrome P450 Reductase, Membrane-Binding Domains 
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 TEI-XML for    default:Reihe_C/55/ZNC-2000-55c-0747.pdf 
 Identifier    ZNC-2000-55c-0747 
 Volume    55 
2Author    Meinrad Boll, LutzW D Weber, Eberhard Becker, Andreas StampflRequires cookie*
 Title    Pathogenesis of Carbon Tetrachloride-Induced Hepatocyte Injury Bioactivation of CC14 by Cytochrome P450 and Effects on Lipid Homeostasis  
 Abstract    The CCl4-induced development of liver damage was studied in monolayer cultures of pri­ mary rat hepatocytes: (1) CC14 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with ß-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triio­ dothyronine had the same effect. (2) CC14 increased the synthesis of fatty acids and triglycer­ ides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CC14 reduced ß-oxidation of fatty acids as assessed by C 0 2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCI4, while saturated fatty acids increased slightly. (5) CC14 exerted a pronounced inhib­ itory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes. 
  Reference    Z. Naturforsch. 56c, 111—121 (2001); received July 28/September 14 2000 
  Published    2001 
  Keywords    Liver Damage, Carbon Tetrachloride, Cytochrome P450 
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 TEI-XML for    default:Reihe_C/56/ZNC-2001-56c-0111.pdf 
 Identifier    ZNC-2001-56c-0111 
 Volume    56 
3Author    D. Ieter, M. Üller-EnochRequires cookie*
 Title    Product Autofluorescence Activation in the Cytochrome P450 Monooxygenase System  
 Abstract    The relative increase of the 7-ethoxycoumarin-O-deethylase and the 6,7-dimethoxycouma-rin-O-demethylase activities were found 93 and 236% using a reconstituted cytochrome P4502B 1 :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 (X.E = 365 nm) or scopoletin (XE = 398 nm), re­ spectively. Addition of the products to the reaction mixtures without irradiation (dark reac­ tion) 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 P 4502B 1-dependent 7-ethoxycoumarin-O-deethylation reaction obtained from Arrhenius plots with and without added umbelliferone and irradia­ tion with XE = 365 nm are 14.7 kJ/mol and 33.5 kJ/mol, respectively, in the temperature range of 27-37 °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 P4502B 1: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. Obvi­ ously the fluorescence light of the products are due for the extremely high activation effect. This is the first report that the products umbelliferone and scopoletin are photoreceptors and phototransducers. 
  Reference    Z. Naturforsch. 49c, 763—771 (1994); received August 1/September 2 1994 
  Published    1994 
  Keywords    Cytochrome P450, Blue Light Activation, Photoreceptor-Phototransducer, Umbelliferone, Scopoletin 
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 TEI-XML for    default:Reihe_C/49/ZNC-1994-49c-0763.pdf 
 Identifier    ZNC-1994-49c-0763 
 Volume    49 
4Author    D. Onald, E. M. O Relan D, T. Hom, J. Fleischm, FrederickT C Orbina, JanisE M CfarlandbRequires cookie*
 Title    Differential Metabolism of the Sulfonylurea Herbicide Prosulfuron (CGA-152005) by Plant Microsomes  
 Abstract    Microsomes isolated from excised shoots of 3-day-old. dark grown, grain sorghum [Sor­ ghum bicolor (L.) Moench, Funk G522DR and DK 41Y] and corn seedlings [Zea mays (L.), Pioneer 3245] metabolized the sulfonylurea herbicide prosulfuron (CGA-152005). Corn microsomes predominantly formed a single major metabolite that resulted from hydroxyla-tion of the phenyl ring at the C5 position. However, sorghum microsomes formed two major metabolites in an approximate 1:1 ratio. One was the 5-hydroxyphenyl metabolite, whereas the second metabolite resulted from ö-demethylation at C4 of the triazine ring. Metabolite identity was established by mass spectrometry and co-chromatography with authentic stan­ dards. Metabolism in both corn and sorghum was greatly enhanced by pretreatment of the seed with naphthalic anhydride and by subirrigation with 2.5% ethanol 24 h prior to harvest. Metabolism required a reduced pyridine nucleotide and was affected by several cytochrome P450 monooxygenase inhibitors (carbon monoxide, tetcyclacis, piperonyl butoxide, 1 amino-benzotriazole, and SKF-525A). The inhibitors differentially affected metabolism of prosul­ furon. Microsomal oxidations from both untreated and inducer-treated tissue responded simi­ larly to the inhibitors. In exploratory studies, microsomes isolated from shoots of wheat [Triticum aestivum L., Pioneer 2548], barley [Hordeum vulgare L., Boone], oats [Avena sativa L., Southern States 76-30-P242] and rice [Oryza sativa L" Gulfmont], and room ripened avocado [Persea americana, Mill., Hass] mesocarp tissue also primarily formed the 5-hydroxy-phenyl metabolite. Titration of seven different avocado microsomal preparations with prosul­ furon provided typical type I difference spectra from which an average binding constant (/Cs) of 187 ± 35 [.im was obtained. Abbreviations: 1-ABT, 1-aminobenzotriazole; alachlor, 2-chloro-.'V-(2.6diethylphenyl)-/V-(methoxymethyl)acet-amide; ALS, acetolactate synthase; CG A 24704, 2-chloro-N-(2,6-dimethylphenyl)-/V-(2-methoxy-l-methylethyl)acet-amide; CGA-150829. 2-amino-4-methoxy-6-methyl-l,3,5-triazine; CGA-152005, prosulfuron, N-[[(4-methoxy-6-methyl-l,3,5-triazin-2-yl) amino]carbonyl]-2-(3,3,3-trifluoropropyl)benzenesulfonamide; CGA -l59902, 2-(3,3,3-tri-fluoropropyl)benzenesulfonamide; CGA-300406, 0-desmethyl prosulfuron, N[[(4-hydroxy-6-methyl-l,3,5-triazin-2-yl)amino]carbonyl]-2-(3,3,3-trifluoropropyl)benzenesulfonamide; CGA-300408, 5-hydroxy prosulfuron, N-[[(4me-thoxy-6-methyl-l,3,5-triazin-2-yl)amino]carbonyl]-2-(3,3,3-trifluoropropyI)-5-hydroxybenzenesulfonamide; chlorsul-furon, l-(2-chlorophenylsulfonyl)-3-(4-methoxy-6-methyl-l,3.5-triazin-2-yl)urea; pCMA, /?-chloro-./V-methylaniline: DMA, /V,/V-dimethylaniline; DMSO. dimethyl sulfoxide; DTT, dithiothreitol; G6P. glucose-6-phosphate; HPLC, high-performance liquid chromatography; LC/ESI/MS. liquid chromatography/ electrospray ionization/mass spectrome­ try; metolachlor, 2-chloro-/V-(2-ethyl-6-methylphenyl)-/V-(2-methoxy-l-methylethyl)acetamide; NA. 1,8-naphthalic anhydride; nicosulfuron, 2-[[(4.6-dimethoxypyrimidin-2-yl)aminocarbonyl]aminosulfonyl]-./V,/V-dimethyl-3-pyridinec-arboxamide; PBO. piperonyl butoxide; primisulfuron. 2-[[[[[4.6-bis(difluoromethoxy)-2pyrimidinyl]amino]carbony-l]amino]sulfonyl]benzoic acid: PVPP. polyvinylpolypyrrolidone; SKF-525A. 2-(diethylamino)ethyl-2.2-diphenylpen-tanoate; tetcyclacis, 5-(4-chlorophenyl)-3,4.5.9,10-pentaazatetracyclo[5.4.102-6,0811] dodeca-3.9-diene; TLC. thin layer chromatography: triasulfuron, l-(2-chloroethoxyphenylsulfonyl)-3-(6-methoxy-4-methyl-l,3.5-triazin-2-yl)urea. Reprint requests to Dr. D. E. Moreland. Telefax: (001) 919-515-7959. 0939-5075/96/0900-0698 $ 06.00 © 1996 Verlag der Zeitschrift für Naturforschung. All rights reserved. D D. E. Moreland et al. ■ M etabolism of Prosulfuron by Plant Microsomes 699 
  Reference    Z. Naturforsch. 51c, 698—710 (1996); received May 14/June 17 1996 
  Published    1996 
  Keywords    Microsomes, Prosulfuron, Cytochrome P450, Mixed Function Oxidases, Herbicide Metabolism 
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 TEI-XML for    default:Reihe_C/51/ZNC-1996-51c-0698.pdf 
 Identifier    ZNC-1996-51c-0698 
 Volume    51 
5Author    Dieter Müller-Enocha, Robert Fintelmann3, Andrey Nicolaevb, Hans GrulerbRequires cookie*
 Title    Enzyme Activity of the Cytochrome P-450 Monooxygenase System in the Presence of Single Chain Lipid Molecules  
 Abstract    (+49) 731-50-23059 and (+49) 7 3 1 -5 0 -2 2 9 8 2 , E-mail: hans.gruler@physik.uni-ulm.de 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 sub­ strate and lipids used for the catalytic center of the protein were obtained: 110 |o,m 7-ethoxy-coumarin (substrate), 1.1 (i m MOG (1-monooleoyl-rac-glycerol), 0.3 | x m SPH (D-sphingosine), 1.5 (!M OA (oleic acid), 3.0 [a m LPC (L-a-lysophosphatidyl-choline), 15.5 [.i m MSG (1-mono-stearoyl-rac-glycerol), 9.5 [i m A A (arachidonic acid), 9.0 [i m PaCar (palmitoyl-L-carnitine), 3.5 [ i m MPG (2-monopalmitoyl-glycerol), 1.5 [i m LPI (L-a-lysophosphatidyl-inositol), 50 [i m LA (lauric acid), 60 [im MA (myristic acid). 85 [im PA (palmitic acid), >100 [tM SA (stearic acid). Only competitive inhibition with the substrate molecule 7-ethoxycoumarin was ob­ served for the single chain lipids LA, MA, PA, SPH, SA, and OA. Non-competitive effects were observed for MPG (-0 .0 3 [i m ~ !) , PaCar (-0 .0 2 [i m _ 1) , MSG (-0 .0 2 3 [ i M ' 1) , LPC (-0 .0 3 [iM _ 1) , A A (-0 .0 3 h m " 1) , and MOG (+0.04 [i m 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. 
  Reference    Z. Naturforsch. 56c, 1082—1090 (2001); received July 11/August 20 2001 
  Published    2001 
  Keywords    Cytochrome P450, Competitive and Non-Competitive Inhibition, Single Chain Lipids 
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 TEI-XML for    default:Reihe_C/56/ZNC-2001-56c-1082.pdf 
 Identifier    ZNC-2001-56c-1082 
 Volume    56