Microsomal fraction, cytochrome

The enzymes responsible for reduction may be located in both the microsomal fraction and the soluble cell fraction. Reductases in the microflora present in the gastrointestinal tract may also have an important role in the reduction of xenobiotics. There are a number of different reductases which can catalyse the reduction of azo and nitro compounds. Thus, in the microsomal fraction, cytochromes P-450 and possibly a flavoprotein are capable of reductase activity. NADPH is required, but the reaction is inhibited by oxygen. FAD alone may also catalyse reduction by acting as an electron donor. 

The microsomal fraction consists mainly of vesicles (microsomes) derived from the endoplasmic reticulum (smooth and rough). It contains cytochrome P450 and NADPH/cytochrome P450 reductase (collectively the microsomal monooxygenase system), carboxylesterases, A-esterases, epoxide hydrolases, glucuronyl transferases, and other enzymes that metabolize xenobiotics. The 105,000 g supernatant contains soluble enzymes such as glutathione-5-trans-ferases, sulfotransferases, and certain esterases. The 11,000 g supernatant contains all of the types of enzyme listed earlier. 

The cytochrome P-450-dependent metabolism of trichloroethylene was studied in hepatic microsomal fractions from 23 different humans (Lipscomb et al. 1997). CYP2E1 was the predominant form of P-450 responsible for the metabolism of trichloroethylene in humans. Incubations of trichloroethylene with the microsomal preparations resulted in hyperbolic plots consistent with Michaelis-Menton kinetics. The values ranged from 12 to 55.7 pM, and were not normally distributed, and the values range from 490 to 3,455 pmol/min/mg protein and were normally distributed. The study authors concluded that the human variability in metabolism of trichloroethylene via P-450-dependent pathways was within a 10-fold range. 

On the other hand, microsomes may also directly oxidize or reduce various substrates. As already mentioned, microsomal oxidation of carbon tetrachloride results in the formation of trichloromethyl free radical and the initiation of lipid peroxidation. The effect of carbon tetrachloride on microsomes has been widely studied in connection with its cytotoxic activity in humans and animals. It has been shown that CCI4 is reduced by cytochrome P-450. For example, by the use of spin-trapping technique, Albani et al. [38] demonstrated the formation of the CCI3 radical in rat liver microsomal fractions and in vivo in rats. McCay et al. [39] found that carbon tetrachloride metabolism to CC13 by rat liver accompanied by the formation of lipid dienyl and lipid peroxydienyl radicals. The incubation of carbon tetrachloride with liver cells resulted in the formation of the C02 free radical (identified as the PBN-CO2 radical spin adduct) in addition to trichoromethyl radical [40]. It was found that glutathione rather than dioxygen is needed for the formation of this additional free radical. The formation of trichloromethyl radical caused the inactivation of hepatic microsomal calcium pump [41]. 

The microsome fractions see Fig. 1) that were prepared from mulberry cortical parenchyma cells were fractionated to 24 or 25 fractions using the 15-50% sucrose linear density gradient centrifugation see Fig. 2). Profiles of the marker enzymes and the protein content are described in Fig. 3. In general, the antimycin A-insensitive cytochrome c reductase activity is exhibited at a lower density than are those of the marker enzymes. The fraction that exhibited the highest antimycin A-insensitive cytochrome c reductase activity for each month was used as the ER-enriched fraction. 

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. 

In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinaoea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPH-cytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4-dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

F3 H activity from the microsomal fraction of cell cultures of C. sinensis (L.) Osbeck cv. Hamlin (Hamlin) has been biochemically characterized and verified to be a cytochrome P450 [113]. The enzyme was shown to use naringenin, dihy-drokaempferol, and kaempferol, but not apigenin as substrates. E3 H activity was also demonstrated from young leaves of Hamlin orange as well as from the flavedo of Hamlin orange, Marsh grapefruit, and Lisbon lemon fruits [113]. The fact... 

A superfamily of heme-dependent monooxygenases that utilize molecular oxygen and NADPH. These enzymes are localized in the endoplasmic reticulum, and are often used as a marker for microsomal fractions obtained upon homogenization of cells. Especially abundant in hver, cytochrome P-450 enzymes play a major role in detoxifi-... 

Microsomal flavin-containing monooxygenases. As well as the cytochromes P-450 MFO system, there is also a system, which uses FAD. This flavin-containing monooxygenase or FMO enzyme system is found particularly in the microsomal fraction of the liver, and the monomer has a molecular weight of around 65,000. Each monomer has one molecule of FAD associated with it. The enzyme may accept electrons from either NADPH or NADH although the former is the preferred cofactor. It also requires molecular oxygen, and the overall reaction is as written for cytochromes P-450 ... 

The most important enzyme involved in bio transformation is cytochrome P-450, which catalyzes many phase 1 reactions. This enzyme is located primarily in the SER (microsomal fraction) of the cell and is especially abundant in liver cells. Cytochrome P-450 primarily catalyzes oxidation reactions and consists of many isoforms (isozymes). These isoenzymes have overlapping substrate specificities. The most important subfamily in humans is CYP3A4, although there is considerable variation in CYP3A4 expression between individuals. 

Ingelmann-Sundberg M, Kaur H, Terelius Y, Persson J-O, Halliwell B (1991) Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Biochem J 276 753-757 Isobe T, Naiki M, Handa S, Taki T (1996) Anal Biochem 236 35-40... 

Benveniste, I., Gabriac, B., and Durst, F., Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction, Biochem. J., 235, 365-373, 1986. 

To measure cytochrome P-450, 10 ml of the 800 g supernatant are centrifuged at 12 000 g for 15 min 5 ml of the supernatant are then centrifuged at 105 000 g for 30 min, after washing twice, microsomes (pellet) are resuspended in phosphate buffer and the protein concentration adjusted to 2 mg/ml. The level of cytochrome P-450 is measured in the microsomal fraction using the method of Omura Sato (1964). 

In order to cover glucuronidation reactions in incubations in microsomal fractions several modifications have been applied in order to optimize conditions. These comprise longer incubation times than necessary for oxidative reactions by cytochrome P450s, and use of modifiers, both to overcome the latency in activity due to the diffusional barriers of the endoplasmatic reticulum (Coughtrie and Fisher 2003 Csala et al. 2004). Modifiers used are detergents or the pore-forming peptide alamethicin (Fisher 2000). Also disruption of cells by sonication is applied (Ethell 1998). 

Most of these studies have established that the enzyme is located in the mitochondrial fraction of tissue homogenates, although the enzyme in the fat body and Malpighian tubules of Locusta is reportedly associated with the microsomal fraction (30). All studies concur that the enzyme is a cytochrome P-450-mediated mixed-function oxidase and its requirements for NADPH and 02 and sensitivity to inhibitors such as carbon monoxide, metyrapone, etc., support this conclusion. As yet, there are no reports as to whether the enzyme is associated with an iron sulfur protein similar to the adrenodoxin of the mammalian mitochondrial steroid... 

The epoxidase catalyzing the 10,11-epoxidation of methyl farnesoate in homogenates of corpora allata from Locusta miqratoria has been studied in detail and has been shown to be a cytochrome P-450-mediated monooxygenase associated with the microsomal fraction. The enzyme is strictly dependent on NADPH and requires oxygen (36). It is sensitive to inhibition by carbon monoxide (36) and to offier compounds such as methyl enedioxyphenyl compounds and imidazoles that are well established inhibitors of the cytochrome P-450-mediated monooxygenases involved in xenobiotic metabolism (37-39). 

NADH-cytochrome 6g reductase was originally solubilized from the microsomes by incubation with cobra venom, and in this form it was thoroughly characterized and its mechanism worked out (17, 307, 308) it has also been solubilized by the action of lysosomes normally contaminating the microsomal fraction (345-347). The two soluble forms were... 

After intravenous treatment of rats with 1200 mg/kg/d CPH for 3 days, homogenates of the renal cortex were separated into subcellular fractions and their protein composition analyzed. The results of the SDS-gel electrophoresis of the renal cortical subtractions showed significant alterations of the polypeptide pattern in the microsomal fraction. The analysis of the polypeptide composition of the microsomal fraction indicated that paralleling to the depletion of cytochrome P-450 isoenzymes in the molecular weight range 50-53,000 was the induction of a polypeptide of molecular weight 44,000 [74]. 

The question rose whether the CPH-induced 44,000 molecular weight polypeptide is a cytochrome P450-isoenzyme. Thus, induction experiments were carried out in which sahne-treated rats were compared with phenobarbital- and CPH-treated rats. Analysis of the polypeptide composition of the microsomal fraction from the phenobarbital group indicated a significant... 

J.L. Fitzpatrick, L.S. Ripp, N.B. Smith, W.M. Pierce Jr., R.A. Prough, Metabolism of DHEA by cytochrome P450 in rat and human liver microsomal fractions. Arch. Biochem. Biophys., 389 (2001) 278. 

It is not so easy to prepare the fungal microsomal fraction and the mixed function oxidases in higher plants. Therefore, the microsomal fraction of rat liver (22) was used instead for the substrate binding assay to ascertain the interaction of (I) and (II) with cytochrome P-450 enzymes. Both (I), (II), and their... 

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