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Nadph-Cytochrome P-450 Reductase

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Nadph-Cytochrome P-450 Reductase STUDIES ON NATIVE AND RECONSTITUTED FMN-DEPLETED NADPH-CYTOCHROME P-450 REDUCTASE CENTRALE LANDBOUWCATALOGUS Y>T'?T y^ "f ^^^ tAND30J"LlS:V :' >ITEIT 0000 0248 7870 WAr;f-NIXOKV Promotor : dr. F. Miiller hoogleraar in de Biochemie fJNO^ljD^ llV°l STELLINGEN 1. Het voorkomen van veel verschillende wijzen van nomenclatuur bij reeds lang bekende iso-enzymen is niet alleen verwarrend voor de betrokken onderzoekers, maar doet ook vermoeden dat andere dan zuiver wetenschappelijke belangen een rol spelen. - W.B. Jacoby, B. Ketterer & B. Mannervik, Bioch.Pharm. 33, (1984), 2539-2540. - T.J. Mantle in Glutathione S-Transferases and Carcinogenesis (1987), ed. T.J. Mantle, C.B. Pickett &J.D . Hayes,Taylo r & Francis London, New York and Philadelphia. 2. De door Wolfes en Pingoud verkregen resultaten met betrekking tot site directed mutagenesis aan Eco Rl zijn niet volledig in overeenstemming met de X-ray resultaten van dit restrictie-enzym van McClarin en Frederick enkunne n scherper geformuleerd worden. - H. Wolfes,A . Pingoud et al., Nucl.Acid.Res. 14,2 2 (1986), 9063. - J. McClarin, C. Frederick, J.M; Rozenberg et al., Science 234 (1986), 1526. 3. De activiteitsbepalingen m.b.v. het gebruik van artificiele i.p.v. natieve electronen acceptoren verdient zeker in het geval van NADPH-cytochroom P-450 reductase meer aandacht. - dit proefschrift. 4. Het feit dat alcohol een bepaalde vorm van cyt P-450 induceert (cyt P-450-alc) en dat deze vorm ook procarcinogenen kan omzetten wijst op toenemendekan s opkanke r bij alcoholgebruik. - J.M. Lasker, J. Rancy, S. Kubota, B.P. Bloswick, M. Black & C.S. Lieber, Biochm.Biophys.Res.Commun. 148 (1987), 232-238. 5. In tegenstelling tot wat Schreuder et al. beweren speelt de C(4) carbonylgroep in het catalytisch mechanisme van PHBH nauwelijks een rol. - H. Schreuder, W. Hoi en J. Drenth InFlavin s and Flavoproteins (1987)eds .D.B . McCormick & D.E. Edmondson, W. de Gruyter Berlin, inpress . 6. De biotechnologische produktie van geneesmidcielen en pesticiden moet kunnen resulteren in een verbod op het zonder meer gebruik van racemische mengsels van deze verbindingen. - Ariens, Science 7, (1986), 200-205. 7. Het door Nisimoto geleverde bewijs dat FAD stevig gebonden is aan FAD-vrij NADPH-cytochroom P-450 reductase wordt niet ondersteund door de resultaten beschreven in hoofdstuk 3va n dit proefschrift. - Y. Nisimoto,J.Biol.Chem . 259, (1984), 2480-2483. - Hoofdstuk 3 van dit proefschrift. 8. Gezien het grote aantal werkzoekenden is het bedroevend orat e zien (horen)ho e er in het algemeen met de belangen van sollicitanten wordt omgesprongen. 9. Specialisten zijn mensen die steeds meer van steeds minder weten. 10. Het isonwaarschijnlij k dat voor het jaar 2000 2D-NMR-spectra, van zelfs eenvoudige eiwitten (meer dan 100 aminozuren), volledig geautomatiseerd geanalyseerd zullen worden. Het blijft voorlopig monnikkenwerk. Studies on native and reconstituted FMN-depleted NADPH-cytochrome P-450 reductase Peter Bonants Wageningen, 18decembe r 1987 fJ/v/03*-O\ ( \\?°! PETER BONANTS STUDIES ON NATIVE AND RECONSTITUTED FMN-DEPLETED NADPH-CYTOCHROME P-450 REDUCTASE Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr. C.C. Oosterlee, inhe t openbaar te verdedigen op vrijdag 18decembe r 1987 des namiddags te vier uur in de aula van de Landbouwuniversiteit te Wageningen. ^ Xbb o\^ Aan mijn ouders Aan Nica VOORWOORD Zoals zovelen voor mij heb ook ik het promotie-onderzoek niet kunnen voltooien zonder de hulp van anderen. Hen wil ik daarvoor met genoegen bedanken. Mijn ouders, die mij in staat stelden om deze lange opleiding te kunnen volgen. Jij Nica, die mij terzijde stond ondanks je onwetend- heid met flavines.Mij n promotor Franz, die op geduldige wijze mijbege - leidde gedurende deze promotieperiode en de laatste maanden, ondanks zijn nieuwe werkkring, toch nog tijd vond om de nodige correcties in het proefschrift aan te brengen. Willem, die er altijd was als je hem nodig had, een onmisbare schakel in de flavine boy's. De andere ledenva n de groep Miillerdi e voor een erg fijne sfeer zorgden waar ik met plezier aan zal terugdenken :Jacques ,Ben , Carlo,Jac ,Ria ,Augus t enWilly , in een eerder stadium. Jacques Vervoort voor zijn hulp in de talloze NMR metingen; Ton Visser voor zijn bereidwillige medewerking in de laser- experimenten enAri e van Hoek voor de experimentele hulp indeze . De studenten die bijmi j een hoofd- danwe l bijvak deden in het kader van hun doctoraalprogramma : Arjen Lommen, Phil Bastiaens,Mario n Rewinkel, Koen Wienk, Margaret Hof, Helga Duivenvoorden en Bertus Welzen. Van hen heb ik zeker het een en ander geleerd. Met name Phil, die met zijn nooit aflatende ijver iedereen verbaasd liet staan,wi l ik bedanken. Jo Haas voor de levering van de voor het onderzoek benodigde levers. Dr.B.Harmsen van de afdeling Biofysische Chemie van de Katholieke Univer- siteit te Nijmegen voor de mogelijkheid om gebruik te maken van de daar aanwezige stopped-flow equipment. Martin Bouwmans voor zijn medewerking bij de tekeningen. Jenny Toppenberg-Fang en in het bijzonder Yvonne Soekhram voor het aanbrengen in dit proefschrift van die letters die mijn vette Ollie niet aankon. En tenslotte Ollie die zonder tegenwerking vele letters tekst verwerkte. CONTENTS Page Voorwoord 5 List of Abbreviations 7 CHAPTER 1 Introduction 8 CHAPTER 2 NADPH-cytochrome P-450reductas e :A 31P NMR study 26 CHAPTER 3 Time-resolved fluorescence spectroscopy onNADPH - 43 cytochrome P-450 reductase CHAPTER 4 Recombination of FMN-depleted NADPH-cytochrome 67 P-450reductas e with different flavins CHAPTER 5 The preparation and some properties of FMN-depleted 85 NADPH-cytochrome P-450 reductase Summary 107 Samenvatting 110 Curriculum Vitae 115 LIST OF ABBREVIATIONS 2'5'ADP :Adenosin e 2'5'-diphosphate 2'AMP :Adenosin e 2'-monophosphate APO :FMN-deplete d NADPH-cytochrome P-450 reductase CD :Circula r Dichroism cDNA :cop y DNA DEAE :Diethy l amino ethyl DTNB : 5,5'-dithio-bis(2-nitrobenzoate) DTT :Dithiothreito l EDTA :Ethylen e diamine tetra acetic acid EPR :Electro n Paramagnetic Resonance FAD :Flavi n Adenine Dinucleotide FMN :Flavi n Mono Nucleotide kDa :kil o Dalton kps :pseud o first order rate constant Xexc :excitatio n wavelength mRNA :messenge r RNA NADPH :Nicotinamid e Adenine Dinucleotide Phosphate, reduced form NADP+ :Nicotinamid e Adenine Dinucleotide Phosphate, oxidized form nm :nanomete r nmole :nanomol e NMR :Nuclea r Magnetic Resonance ns :nanosecon d OX :Oxidize d PAGE :Polyacrylamid e gelelectrophoresls PC :Phosphatidylcholin e PCMB :p-hydroxychloromercur i benzoate PMSF :Pheny l methane sulphonyl fluoride PPM :part s per million REC :FM N reconstituted FMN-depleted NADPH-cytochrome P-450 reductase SDS :Sodiu m dodecyl sulphate SQ :Semiquinon e (one-electron reduced) TLC :Thin-laye r chromatography TNBS :Sodiu m 2,4,6-tri-nitrobezene sulfonate UC :Ultracentrifugatio n UV :Ultraviole t photolysis © e H or OH pH<7 pH>9 "2 z» 52- CH2— ICHOHlj— CH20- P—O-pP—0 —C riboflavin Ivitamh 02' riboflavin - 5'- monophosphate IFMN) flavin- adenine - dmucleotkje (FADI FIGURE1 Structures of some common flavins CHAPTER 1 INTRODUCTION Innatur e there are an enormous number of enzymes active in various species. This thesis deals with one of those enzymes , namely NADPH- cytochrome P-450 reductase. This enzyme ispar t of a group of enzymes called flavoproteins , which contain one or more flavins.Th e flavin is a prosthetic group which is covalently or non-covalently bound to enzy­ mes and has a specific function in the overall activity of the enzyme in question. Fig. 1show s the most common flavin cofactors which are derived from vitamine B2- NADPH-cytochrome P-450 reductase ispresen t particular­ ly in mammalian liver microsomes . It is an unusual membrane-bound flavoprotein because it contains two different flavins : FAD (Flavin Adenine Dinucleotide) and FMN (Flavin Mono Nucleotide) in one polypep­ tide chain. History: The first report on this enzyme was made by Horecker in 1949 [1]. He reported on an NADPH-mediated cytochrome creductas e in animal tissue which contained only one flavin (FAD). Despite the excellent work, the physiological role of the enzyme and the localization of the enzyme in the tissue remained unclear. However at that time one could not foresee a possible function for this flavoprotein in microsomal electron trans- port. In the mid 1950's Strittmatter and Velick [2]observe d in their studies a different microsomal fraction which reduced cytochrome c 20 times faster in the presence of NADPH rather with NADH as electron donor. They suggested that this activity might be the same as that described by Horecker. In 1962 Williams and Kamin [3], and shortly thereafter Phillips and Langdon [4], showed that NADPH-cytochrome c reductase was localized in the microsomes. Both groups speculated on the possible role of this flavoprotein in aromatic and steroid hydroxylations , and drug demethylations .Phillip s and Langdon [4]presente d some evidence for the participation of this enzyme in amicrosoma l electron transport system not involving cytochrome c. In the mean time Williams [5]mad e the disco­ very of a carbon monoxide-binding pigment in rat liver microsomes exhibiting an intense absorption at 450n m in its reduced carbon monoxide bound form .Omur a and Sato [6]prove d in 1962 that this protein contains heme and studied the heme protein inmor e detail. In 1963 and 1964 itwa s shown by Estabrook [7]an d Cooper [8]tha t this cytochrome P-450 was involved in agrea t variety of hydroxylation and oxidative dealkylation reactions. The selective increase of both cytochrome P-450 and NADPH- cytochrome P-450reductas e inmicrosome s from drug-treated animals was a first indication that the reductase was also involved in drug and steroid metabolism [9-12].
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