Liver cell microsomal fractions

Figure 7.2 Synthesis of HOTYR glucuronides using liver cell microsomal fractions...

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

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]. 

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). 

NMOR is mutagenic in bacterial assays in the presence of activated liver microsomal fractions. However, NMOR did not induce DNA damage in either human or rat kidney cells in vitro as determined by DNA strand breakage. ... 

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. 

If liver cells (hepatocytes) are isolated and grown in culture, drugs are exposed to a similar array of enzymes. Because CYP enzymes are bound to the internal membrane fraction of hepatocytes, the liver can be homogenized and a preparation of vesicles of the hepatocyte endoplasmic reticulum called microsomes can be incubated with drug molecules. This preparation suffers because many of the soluble Phase II conjugation enzymes that are found in the cellular cytoplasm are lost. An alternative method for measuring microsomal metabolism involves isolation of the so-called S9 fraction , which includes the cytosolic soluble conjugation enzymes. 

Vitamin K is critical to (he formation of clotting factors VII. IX. and X. These factors are glycoproteins that h.avc y-vaiboxyglutamic acid residues at the N-terminal end of (he ptptide chain. The enzyme involved in forming an active prothrombin is a vitamin K-dcpendcn( carboxyla.se located in the microsomal fraction of liver cells. It has been sug-... 

The absorption of natural vitamin K from-the small intestine into the lymphatic system is facilitated by bile, as is true for other fat-soluble materials. Efibciency of absorption varies from 15% to 65% as reflected by recovery in lymph within 24 hours. Vitamins Ki and K2 are bound to chylomicrons for transport from mucosal cells to the liver. Menadione (Ks) is more rapidly and completely absorbed from the gut before entering the portal blood. In liver, intraceUular distribution is mostly in the microsomal fraction, where phenylation of menadione to form K2 occurs. Release of vitamin K to the blood stream allows association with circulating P-lipoproteins for transport to other tissue. Significant levels of vitamin K have been noted in the spleen and skeletal muscle. 

The intracellular distribution of copper has been studied. A distinct pattern of distribution was foimd among the four subcellular fractions that can be separated by differential centrifugation. In rat liver, 64.3% of the total copper is found in the soluble fraction, 8.2% and 5.0% in the mitochondrial and microsomal fractions, respectively, and 20.3% in the fraction containing nuclei and cell residue. These results obtained by Thiers and Vallee (T2) were confirmed by Hermann and Kun (HIO). In the livers obtained at autopsy of two adult men. Porter has found a similar subcellular distribution of copper (Pll, P15). 

The metabolism of PAHs has been studied extensively in vitro and in vivo. The most commonly used system is the rat liver microsomal fraction, although other species are also used. Cells and cultured tissues from human and other animals have also significantly contributed to the elucidation of the PAH metabolic scheme. 

These are major enzyme systems involved in phase I reactions. Localized in the smooth endo-plastic reticulum (microsomal fraction) of cells (especially liver, but including GI tract, lungs, and kidney). 

ACAT may also be activated by covalent phosphorylation. Studies with microsomal ACAT that was incubated with Mg " " and ATP showed an increase in activity, and a further increment in activity upon addition of a supernatant fraction from liver cells [32]. This type of activation seems similar to that observed with HMG-CoA reductase [33]. 

Ascorbic acid within the cells was localized in the cytoplasm (G3). More recent studies in rat liver and ox adrenals by the method of differential centrifugation have shown that most of the ascorbic acid was in the soluble and microsomal fractions (G2). The sum of ascorbic acid in the different fractions exceeded that of the whole tissue analyses by 40 %, suggesting to these workers the existence of bound ascorbic acid not liberated by extraction of the whole tissue. The fact of some localization in microsomes deserves confirmation. 

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