Microsomal fractions

Vane and co-workers isolated a new prostaglandin (initially called PGX) from microsomal fraction of stomach. The structure was established by chemical synthesis from PGP2a (Ref. 1). 

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 five enzyme activities are localized in the microsomal fraction in rat testes, and there is a close functional association between the activities of 3P-OHSD and A -isomerase and between those of a 17oc-hydroxylase and 17,20-lyase. These enzyme pairs, both contained in a single protein, are shown in the general reaction sequence in Figure 42-5. 

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. 

Polyphenols and flavanoids in rat liver microsomal fractions have been demonstrated to inhibit glucuronidation of estrone and estradiol in vitro (Zhu et al, 1998). In addition, flavonoids have also been found to induce phase I and II enzymes in rats including UDP-glucuronosyl transferase (Seiss et al, 1996). However, the effects of phytoestrogens have not been evaluated for either their inhibition or induction of glucuronosyl transferase activity. 

The second study was performed using either cytosolic or microsomal fractions from rat liver as the in vitro metabolic mammal models [238]. The studied compound, benzofuroxan (128, Fig. 20), is metabolized to o-quinone dioxime and 2,3-diaminophenazine (Scheme 4). 

Albano, E., Lott, K.A.K., Slater, T.F., Stier, A., Symons, M.C.R.and Tomasi, A. (1982). Spin trapping studies on the free radical products formed by metabolic activation of carbon tetrachloride in rat liver microsomal fractions, isolated hepato-cytes and in vivo in the rat. Biochem. J. 204, 593-603. 

An elegant study of lycopene uptake in LNCaP, PC-3, and DU-145 cells using beadlet-delivered 1.48 J.M all-trans lycopene (a maximal level in human plasma) found that all three cell lines rapidly took up lycopene during the first 10 h of incubation. Cells continued to accrue lycopene, but more slowly, over the next 48h. The uptake by the LNCaP cells was 2.5-fold higher than PC-3 cells and 4.5-fold higher than DU-145 cells at 24h of incubation but lycopene showed no affinity for the AR receptor, which is expressed in the LNCaP cells (Liu et al. 2006). LNCaP uptake followed Michaelis-Menten kinetics with a V m i, of 66.3pmol/106 cells/h and a Km of 7.72 pM lycopene. Because of the sensitivity of their LC-MS-MS lycopene assay, Liu et al. were also able to investigate the subcellular lycopene distribution. The nuclear membrane contained 55%, the nuclear matrix 26%, and the microsomal fraction 19% of the intracellular fraction. The cytosol contained no lycopene(Liu et al. 2006). 

Nitropyrene. 1-Nitropyrene is the principal nitro PAH found in diesel exhaust (40) and, therefore, has been the subject of intense study. Nachtman and Wei (133) found that under anaerobic conditions, 1-nitropyrene was reduced by hepatic S9, cytosol or microsomes to principally 1-aminopyrene. Only limited reduction occurred in the absence of cofactors, while maximum metabolism was observed in the presence of both FMN and NADPH. Although the microsomal fraction had the greatest specific activity toward 1-nitropyrene metabolism, the cytosol had 30 times the total activity. 

The enzyme involved in the degradation of the dyes has been shown to be azoreductase. The enzymes were first isolated from the intestinal microflora and was later found to be produced by the cytosolic and microsomal fractions of the liver [47]. The enzyme was sensitive to oxygen and was inactivated by oxygen. In experiments involving intestinal anaerobic bacteria, Rafii et al. found the requirement of... 

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 NADH- and oxygen-dependent microsomal metabolism of the di-, tri- and tetraethyl substituted derivatives of germanium, tin and lead was shown to give rise to ethylene as a major product and ethane as a minor product27. These reactions were shown to be catalyzed by the liver microsomal fractions. 

Mehta RR, DasGupta TK (1987) Antiestrogen binding sites in microsomal fractions of malignant and nonmalignanthuman breast tissues. Breast Cancer Res Treat 9( l) 61-67... 

It was reported that the distribution and activities of esterases that catalyze pyrethroid metabolism using several human and rat tissues, including small intestine, liver, and serum, were examined [30]. The major esterase in human intestine was hCE2. //c/n.v-Permethrin was effectively hydrolyzed by pooled human intestinal microsomes (five individuals), while deltamethrin and bioresmethrin were not. This result correlated well with the substrate specificity of recombinant hCE2. In contrast, pooled rat intestinal microsomes (five animals) hydrolyzed trans-permethrin 4.5 times slower than the human intestinal microsomes. Furthermore, pooled samples of cytosol from human or rat liver were ca. half as hydrolytically active as the corresponding microsome fraction toward pyrethroids however, the cytosolic fractions had significant amounts (ca. 40%) of the total hydrolytic activity. Moreover, a sixfold interindividual variation in hCEl protein expression in human hepatic cytosols was observed. 

Collect the supernatant and then ultracentrifuge at 200,000g for 20 min to precipitate the crude microsomal fraction that contains the ER, Golgi complex, tonoplast, and plasma membrane. 

Reultracentrifuge the resuspended crude microsome fraction at 200,000 for 20 min to exchange the buffer system. 

To suspend a pellet of microsomal fraction, we used a glass pipet with a bent tip and a Teflon/glass homogenizer. 

Most of the intact mitochondria and chloroplasts may be removed at the first centrifugation. However, a large amount of these organelles still existed in the microsomal fraction as estimated from the results of marker enzyme assays. 

Overlay 4 mL of crude microsomal fraction onto the linear sucrose density gradient solution. 

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. 

Fig. 1. Flow chart for isolation of endoplasmic reticulum from mulberry cortical parenchyma cells. All procedures are performed at 4°C. Centrifugation times are given as the time at full speed, not included the acceleration and braking time. Resuspension of the microsomal fraction pellet is accomplished using a Teflon/ glass homogenizer.

Fig. 4. Localization of WAP27 and WAP20 in the crude microsome fractions and the relation with marker-enzyme activities in three organelles (ER, tonoplast, and Golgi). SDS-PAGE of fractionated proteins by isopycnic linear sucrose density gradient centrifugation of microsome fraction of mulberry cortical parenchyma cells was performed using 6-pL samples in each fraction. Immunoblot analysis was performed with anti-WAP27 and anti-WAP20 antibodies. (From ref. [1], with permission from the American Society of Plant Physiologists.)...

Typically, microsomal fractions are used for TDI analysis, but in many cases TDI is performed in rCYPs. The ability to use rCYPs for TDI relies on the accurate ability to extrapolate the rCYP data [186]. The second requirement for successful extrapolation of rCYP data is that the clearance pathways of the inhibitor in question are understood. For example, raloxifene has been shown to be a strong MBI of CYP3A4 in microsomes and rCYPs, but in hepatocytes the most predominant pathway is glucuronadation, which would effectively mask any CYP3A4 inactivation observed in... 

Remove the supernatant fluid. This is the soluble fraction. Resuspend the deposit in the same volume of buffer as before (Xml). This is the microsomal fraction. 

The microsomal fraction was first obtained by Claude in 1943. In addition to lipid in the fraction, he noted the presence of RNA-rich granules, consistent with reports from Brachet that cytoplasm stained for RNA by the methyl-green/pyronin procedure. Glucose-6-phos-phatase was a prominent enzyme when the fraction was prepared from liver. Since density gradient sedimentation showed G-6-P-ase was absent from mitochondria and lysosomes, it was used as a marker for liver microsomes. 

Literature data on cytotoxic effects of photoexcited fullerene C60 are controversial. In the studies on transformed B-lymphocytes of Raji fine, phototoxic action of water-soluble carboxy-C60 was not revealed even upon its concentration of 5 x 10 5 M (Irie et al., 1996). In the study (Kamat et al., 2000) damaging effect of fullerenes C60 in dependence on intensity of irradiation toward CHO cells has been demonstrated. Using microsomal fraction of rat liver that was treated with C -cyclodextrin complex, it was shown that already in 5-30 min after UV-irradiation the accumulation of LPO products occurs that is suppressed by antioxidants like ascorbic acid and a-tocopherol. Similar effect of fullerenes C60 has been revealed in microsomal fraction of the cells of ascitic sarcoma 180 (Kamat et al., 2000). 

The FAS multi-enzyme complex synthesizes saturated C16 fatty acids, but cells and tissues need unsaturated and longer chain fatty acids. The palmitoyl-CoA can be modified by either chain elongation and/or oxidation in order to produce different fatty acid molecules. Both elongation and desaturation occur within the smooth endoplasmic reticulum (SER, microsomal fraction) of the cell. 

To acquire information on the intrinsic metabolic activity of aquatic organisms, liver of carp (Cyprinus carpio Linnaeus), rainbow trout (Salmo gairdneri) and freshwater snail (Cipango-paludina japonica Martens) was dissected out, homogenized in 0.1M phosphate buffer, pH 7.5, and centrifuged at 105,000 g for 60 min to obtain the microsome-equivalent (described as the microsomal fraction hereafter) fraction. The protein content of microsomal and submicrosomal (supernatant fractions by Lowry s method, microsomal P-450 content ( ), activity of aniline hydroxylase (4) and aminopyrine N-demethylase (5) were determined. 

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