Monooxygenase reconstituted

Karuzina, I.I., VG. Zgoda, G.P Kuznetsova, N.F. Samenkova, and A.I. Archakov (1999). Heme and apoprotein modification of cytochrome P450 2B4 during its oxidative inactivation in monooxygenase reconstituted system. Free Radio. Biol. Med. 26, 620-632. 

DMN oxidative demethylation has been shown to be a liver mi-crosome cytochrome P-450 monooxygenase (10) Lotlikar et al. (11) found that a reconstituted enzyme system, consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidyl choline was effective in catalyzing the demethylation of DMN. The most commonly accepted mechanism for the oxidative demethylation of DMN and, by extension, of other dialkyInltrosamlnes is shown in Scheme 1. 

Adults injected ip for 3 days at 80 mg/kg BW daily then killed 24 h after last injection Heme destruction in the P-448-containing reconstituted monooxygenase system reactive epoxide, or possibly nonepoxide, intermediate metabolites may participate in cytochrome P-448 destruction 24... 

Mueller and Miller (33) and Brodie et al. (34) were the first to show that enzymes in the microsomal fraction of rat liver could effectively oxidize xenobiotics. Comparable enzymes (aryl hydrocarbon monooxygenases) were later reported in the hepatic tissues of fresh water and marine fish by Creaven et al. (35) and Buhler and Rasmusson (36). Reconstituted hepatic microsomal systems require cytochrome P-450 for monooxygenase activity in both mammals (37) and fish (38,39). Bend et al. 

Selected entries from Methods in Enzymology [vol, page(s)] Activity in ethanol oxidation, 233, 118 in hydroxyethyl radical formation analysis with reconstituted vesicles, 233, 127 characterization, 233, 123-125 monooxygenase activity, 231, 574-575 reductase [hemoglobin-catalyzed reactions with, 231, 573-574 oxygen concentration and, 231, 579 pH dependence, 231, 580 reaction mixtures, 231, 578 oxygen content, measurement, 231, 587-588 reductase concentration and, 231, 580 time dependence, 231, 578 preparation, 231, 577]. 

PARATHION METABOLISM BY A RECONSTITUTED MONOOXYGENASE SYSTEM FROM RABBIT LIVER ... 

We have also examined the metabolism of parathion by purified reconstituted monooxygenase systems isolated from the livers of... 

Figure 4. Linearity of the metabolism of parathion and benzphetamine by a reconstituted monooxygenase oxidase enzyme system from rabbit liver. The 0.5-mL reaction mixture contained 50 fig of sodium deoxycholate, 15 iig of dilauroyl l-5-phosphatidylcholine, 1.5 units of NADPH-Cytochrome c reductase, 0.5 nmol of Cytochrome P-450, 0.05M Hepes buffer (pH 7.8), 0.015M MgCh, O.lmU EDTA, and 5 X lO M [ethyl- C] parathion or / X 10 M benzphetamine.

Figure 6. Elution profile of protein, radioactivity, and thiocyanate from a Sepha-dex G-25 column of reconstituted monooxygenase system from rat liver that had been incubated with [ 5] parathion. The 5-mL incubation mixture contained 20 nmol Cytochrome P-450 (specific activity 16.4 nmol/mg protein), 5 units NADPH-Cytochrome c reductase, 600 fig dilauroyl L-3-phosphatidylchoUne, 600 fig sodium deoxycholate, and 1 X IO M p 5] parathion. The remainder of the incubation mixture is described in Figure 4. The incubation time was 5 min. One-milliliter fractions were collected. The radioactivity (x) represents cpm/0.1 mL. The OOggo (o) was measured on each 1-mL fraction (20).

Additional work in our laboratory using a reconstituted monooxy-genase system containing purified cytochrome P-450 has indicated that the sulfur atom released from parathion covalently binds to at least three other, as yet unidentified, amino acids on the cytochrome P-450 molecule. It appears to be clear that the binding of the sulfur atom to cysteine or cysteines is responsible for the loss of cytochrome P-450 detecteable as its CO complex and the accompanying loss of monooxygenase activity (25). 

Incubation of l,l,2,2,-tetrachloro[l,2- 4C]ethane with a reconstituted monooxygenase system or with intact rat liver microsomes led to the formation of a metabolite capable of binding covalently to proteins and other nucleophiles. The only soluble metabolite detected upon incubation of 1,1,2,2-tetrachloroethane with a reconstituted system was dichloroacetic acid. Pronase digestion of the C-labcllcd microsomal proteins indicated the presence of several derivatized amino acids, which were hydrolysed by alkali to yield dichloroacetic acid. The results are consistent with biotransformation of 1,1,2,2-tetrachloroethane by cytochrome P450 to dichloroacetyl chloride, which can bind covalently to various nucleophiles or hydrolyse to dichloroacetic acid (Halpert, 1982). 

Pseudomonas oleovorans contains P. oleovorans monooxygenase (POM), which is a typical co-hydroxylase for hydroxylation of the terminal methyl of alkanes as well as epoxidation of terminal olefins. The co-hydroxylation system of P. oleovorans was reconstituted from purified components, POM, rubredoxin, and a flavoprotein reductase [122], In the presence of NADH and oxygen, it oxidizes a wide range of aliphatic methyl alkyl sulfides. Enantioselectivities are very much dependent of the length of the alkyl chain of Me-S(0)-R, as exemplified by the following results ... 

The relative contribution, in intact microsomal preparations, of the two monooxygenases to the formation of N-oxygenated C=N functionalities has been frequently assessed by measurements in the presence and absence of selective enzyme inhibitors or positive effectors. These observations were supplemented by studies using highly purified native or recombinant proteins in reconstituted systems. 

An approach for determining the presence of multiple monooxygenases in rat liver microsomes was the use of chemicals that selectively affected certain monooxygenase activities but not others. For example, 7,8-benzoflavone (a-naphthoflavonc) markedly inhibited the hydroxylationofbenzo[a]pyrene by liver microsomes from 3-methylcholanthrene treated rats or by a cytochrome P448-dependent reconstituted enzyme system (68,69), but there was no effect or only a small stimulatory effect of 7,8-benzoflavone on benzo[a]pyrene metabolism in livers from untreated rats (68). 

Shimada T, Imai Y, Sato R. 1981. Covalent binding of polychlorinated biphenyls to proteins by reconstituted monooxygenase system containing cytochrome P-450. Chem Biol Interact 38 29. ... 

Both electron transfer chains cannot substitute each other in reconstituted systems, which points to a different phylogenetic origin of mitochondrial and bacterial monooxygenases on the one hand and the microsomal enzymes on the other. Microsomes also contain cytochrome bs which is able to donate electrons to the monooxygenases. However, the physiological role of this cytochrome is still controversial and more than one function has to be considered. 

The first monooxygenase system isolated was the steroid-11/3-monooxygenase from adrenal mitochondriaIn a reconstituted system the heme-sulfiir protein required an iron-sulfur protein, a flavoprotein and NADPH for reduction and dioxygen acti-... 

In the reconstituted systems the reductase proved not to be specific for a particular cytochrome P450. The enzyme from liver could reduce the to-monooxygenase from kidney and vice versa and its antibody was effective in inhibiting the NADPH-mediated cytochrome c reduction in spleen microsomes. 

James MO, Khan MAQ, Bend JR (1979b) Hepatic microsomal mixed-function oxidase activities in several marine species common to coastal Florida. Comp Biochem Physiol 62C 155-164 James MO, Sherman B, Fisher SA, Bend JR (1982) Benzo(a)pyrene metabolism in reconstituted monooxygenase systems containing cytochrome P-450 from lobster Homarus americanus) hepatopancreas fractions and N ADPH-cytochrome P-450 reductase from pig liver. Bull Mt Desert Isl Biol Lab 22 37-39... 

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