Microsomes turnover

Introduction of R2 = NH2 into the structure of the parent imine presented in equation 4 yields an amidine, which can be subject to N-oxygenation. Thus, a series of benza-midines have been detected to undergo conversion to benzamidoximes (24a), and this process appears to be catalyzed by P-450, as evidenced by susceptibility of the microsomal turnover to CO or SKF 525A and the inability of highly purified hog liver FMO... 

It is interesting to note that all the known direct effects of protein S3Tithesis on RNA turnover are concerned rather specifically with individual nucleotides, rather than with RNA synthesis or turnover as a whole. It is possible that all of the synthesis and turnover of RNA observed in vivo in connection with protein synthesis is of this type, at least in the cytoplasm. Although there seems to be a limited turnover of the microsomal RNA 393), there is no certainty on this point. It is possible, for example, that the labeling observed in vivo is due to synthesis of complete new particles, rather than to the turnover of a small fraction of the RNA of all the particles. At any rate, it is quite certain that microsomal turnover is small compared with that of the cytoplasmic RNA. 

During catabolic and anabolic processes, a renovation of the molecular cellular components takes place. It should be emphasized that the catabolic and anabolic pathways are independent of each other. Be these pathways coincident and differing in the cycle direction only, the metabolism would have been side-tracked to the so-called useless, or futile, cycles. Such cycles arise in pathology, where a useless turnover of metabolites may occur. To avoid this undesirable contingency, the synthetic and degradative routes in the cell are most commonly separated in space. For example, the oxidation of fatty acids occurs in the mitochondria, while the synthesis thereof proceeds extramitochondrially, in the microsomes. 

If a high turnover (>50%) is observed in the initial screening incubation at 10-50 iM, then a higher substrate concentration should be tested to obtain a good balance between the enzyme productivity and the conversion yield. The substrate concentration should be mainly selected based on the activity of enzymes, and may be increased until the benefit of enzyme productivity increase is offset by cost and availability of the substrate. The solubility of substrate in buffer may limit the use of high concentrations, but a low aqueous solubility compound is readily absorbed by microsomes/S9, which will help to keep the substrate in the incubation mixture even at a much higher concentration than its buffer solubility. Substrate concentration up to sub-millimole level can be used as long as no inhibitory effect is shown and a reasonable turnover is achieved. 

The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b, and cytochrome P-1+50 in the rat. Biochim. Biophys. Acta (1970) 201, 20-05-... 

M. Arandt, F. Muller, A. Mecky, W. Hinz, P. Urban, D. Pompon, R. Kellner, F. Oesch, Catalytic Triad of Microsomal Epoxide Hydrolase Replacement of Glu404 with Asp Leads to a Strongly Increased Turnover Rate , Biochem. J. 1999, 337, 37 - 43 M. Arandt, H. Wagner, F. Oesch, Asp333, Asp495, and His523 form the Catalytic Triad of Rat Soluble Epoxide Hydrolase , J. Biol. Chem. 1996, 271, 4223 - 4229. 

No studies were located indicating that any populations are unusually susceptible to heptachlor or heptachlor epoxide. There is a possibility that very young children may exhibit particular susceptibility to hepatic effects because of the immaturity of the hepatic microsomal system. Heptachlor is bioactivated to produce heptachlor epoxide which is more toxic than heptachlor. Preadolescent children have a greater rate of glutathione turnover, and they are expected to be more susceptible to heptachlor epoxide-induced toxicity. Their susceptibility would probably depend upon their ability to detoxify heptachlor epoxide. Individuals who show reduced liver function for other... 

The classic hard nucleophile used in trapping hard metabolite electrophiles is cyanide. Indeed, with modern detection sensitivities it is now often possible to detect cyanide adducts from microsomal incubations that were quenched with acetonitrile, with the residual cyanide in the acetonitrile reacting with the electrophilic species. In an experiment designed specifically to generate and detect cyanide adducts, millimolar concentrations of cyanide may be included in a microsomal incubation with no detrimental effect on the metabolic turnover. 

Use a system which allows the preparation of subcellular fractions. Microsome preparation provides a 5- to 10-fold enrichment in cytochrome P450 expression levels and also permits longer incubation times. This enrichment and concomitant increase in metabolizing capacity per unit volume may be important in detecting metabolism of slow turnover substrates. 

CYP2C8 has recently been established at the principal enzyme responsible for the 6a-hydroxylation of taxol, the principal route of metabolism for this anticancer drug (Rahman et al., 1994). For this study, a combined approach of correlation analysis with levels of immunoquantitated CYP2C8 protein in human liver microsomes, inhibition analyses in human liver microsomes, and vv-expressed proteins established that CYP2C8 was the principal enzyme for taxol 6a-hydroxylation and that this enzyme catalysed the reaction at high turnover (30/min). 

CYP3A4 has been expressed in human lymphoblasts (Crespi et al., 1991a). The expression level in this report was quite low. Recent modifications to the promoter for cDNA expression and coexpression of OR have led to a 40-fold increase in catalytic activity. The mean testosterone 6j8-hydroxylase activity in microsomes from h3A4/OR cells (cultured in the presence of dexamethasone to induce cytochrome P450 reductase activity) [1000 pmol/(mg min)] is comparable to the mean values observed in human liver microsomes [1070 pmol/(mg min) Yamazaki et al., 1993]. The turnover number for testosterone and CYP3A4 in the human lymphoblasts was 15/min for endogenous OR levels and increased to 44/min with OR coexpression. 

Purified CYP2E1 from mouse and rat liver microsomes in a reconstituted system is a very active catalyst of dimethylformamide oxidation, the turnover being about 10 mnol/ min per nmol P450 for both species however, the affinities are very different. The values for mouse and rat CYP2E1, respectively, were about 0.08 mM and 1.1 mM (Chieli etal., 1995). 

Further support for the hypothesis that Ca2+ plays a central role in regulating phytoalexin accumulation is provided by experiments in which the turnover of phosphatidylinositol was measured in the plasma membrane of elicitor-treated carrot cells [17]. The carrot cells were first labelled with [3H]myo-inositol and, after the addition of elicitors, acid extracts of the cells were analyzed chromatographically for the production of inositol trisphosphate (IP3). In cells treated with elicitor, the release of radioactive IP3 increased with time and attained a maximum at 3 - 5 min after treatment. Phospholipase activity responsible for the degradation of phosphorylated phosphatidylinositol increased correspondingly. Several reports have shown that IP3 induces rapid release of Ca2+ from intracellular stores in animal cells [18, 19]. Studies on plant cells have also demonstrated that exogenous IP3 releases Ca2+ from microsomal preparations at micromolar concentrations, although only limited... 

SERCA pumps in cultured COS cells. Their functional properties were studied using isolated microsomes. The initial in vitro studies reported that SERCA1 and SERCA2a isoforms shared similar Ca2+ affinity and velocity of Ca2+ uptake (Vmax). Subsequently, a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform (5.0- versus 2.6-fold increase in calcium uptake rate) (Sumbilla et al 1999). 

Figure 8 Turnover of DFB to DFH in microsomes prepared from cell lines expressing a single CYP. Abbreviations DFB, [3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methyl-sulfonyl)phenyl]furan-2(5//)-one] DFH, [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl) phenyl] furan-2(5//)-one] CYP, cytochrome P450. Source From Ref. 32.

Supersomes are available containing cDNA-expressed cytochrome b5 or not. Since insect cell microsomes do not contain significant amounts of cytochrome b5 the incorporation of this enzyme increases the respective cytochrome P450 activity which is of importance if the substrate specific turnover is increased. 

Various other heme-peroxidases were found to catalyze the enantioselective sulfoxidation of alkyl aryl sulfides. These included horseradish peroxidase (HRP),34,35 cytochrome c peroxidase (CcP),36 microsome peroxidase (MP),37 lac-toperoxidase (LPO),38 and dioxygenase.39 However, their turnover numbers (TON) and enantioselectivities were much lower than those observed with CPO (Table 4). 

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