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PubMed1997

Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation.

The effect of fatty acid binding proteins (FABPs) on two key steps of microsomal phosphatidic acid formation was examined. Rat liver microsomes were purified by size-exclusion chromatography to remove endogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to avoid cross-contamination with such proteins from native tissue. Neither rat liver (L-FABP) nor rat intestinal fatty acid binding protein (I-FABP) stimulated liver microsomal fatty acyl-CoA synthase. In contrast, L-FABP and I-FABP enhanced microsomal conversion of [14C]oleoyl-CoA and glycerol 3-phosphate to [14C]phosphatidic acid by 18- and 7-fold, respectively. The mechanism for this stimulation, especially by I-FABP, is not known. However, several observations presented here suggest that, like L-FABP, I-FABP may interact with fatty acyl-CoA and thereby stimulate enzyme activity. First, I-FABP decreased microsomal membrane-bound oleoyl-CoA. Second, oleoyl-CoA displaced I-FABP bound fluorescent fatty acid, cis-parinaric acid, with Ki of 5.3 microM and 1.1 sites. Third, oleoyl-CoA decreased I-FABP tryptophan fluorescence with a Kd of 4.2 microM. Fourth, oleoyl-CoA red shifted emission spectra of acrylodated I-FABP, a sensitive marker of I-FABP interactions with ligands. In summary, the results demonstrate for the first time that both L-FABP and I-FABP stimulate liver microsomal phosphatidic acid formation by enhancing synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate.

Acyl Coenzyme A↗
PubMed1994

Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C]dextromethorphan.

The activity of human liver microsomal cytochrome P4502D6 (CYP2D6) is readily estimated by following the O-demethylation of [O-methyl-14C]dextromethorphan. The basis of the assay is the quantitative measurement of [14C]formaldehyde (0.05-4.0 microM) after addition of NaOH to the microsomal incubates and extraction with methylene chloride. The assay is relatively simple, sensitive (limit of detection is approximately 5.0 pmol HCHO/h/mg microsomal protein) and does not require the use of HPLC or an internal standard. Formation of radiolabeled formaldehyde in human liver microsomes is linear for 20 min, up to a final protein concentration of 1.0 mg/ml. Furthermore, the O-demethylase activity in a panel of microsomes prepared from a series of human livers was significantly correlated with the immunochemically determined levels of CYP2D6 protein (r = 0.925, p < 0.001), and was inhibited (> 89%) by quinidine and lobeline. In addition, [O-methyl-14C]-dextromethorphan O-demethylation was exclusively catalyzed by cDNA-expressed CYP2D6 in microsomes prepared from human B-lymphoblast cells. The method is suitable for rapid screening of compounds as potential CYP2D6 cosubstrates and/or inhibitors.

B-Lymphocytes↗
PubMed1994

Induction of the cytochrome P450 3A subfamily in rat liver correlates with the binding of inducers to a microsomal protein.

The specific binding of the archetypal cytochrome P450 3A subfamily (CYP3A) inducer dexamethasone is examined in microsomal fractions since rat and human liver CYP3A is induced by glucocorticoids through a mechanism which is apparently independent of the cytosolic glucocorticoid receptor. Dexamethasone binds in a specific and saturable manner to microsomes with an affinity constant (Kd) of 59 +/- 12.9 nM which compares to a Kd of 2.3 +/- 0.17 nM in cytosol. The total receptor concentrations ([LR]emax) in microsomes and cytosol are 9.5 +/- 1.67 pmols/mg protein and 410 +/- 167 fmol/mg protein respectively. The microsomal binding of dexamethasone is antagonised by several transcriptional and/or post-transcriptional CYP3A inducers with decreasing potency pregnenolone 16 alpha carbonitrile > metyrapone > phenobarbitone. Troleandomycin, which indirectly induces CYP3A1 in vivo and by protein stabilisation, does not antagonise the binding of dexamethasone in microsomes. The transcriptional and/or post-transcriptional induction of CYP3A may therefore be associated with the interaction of inducers with a microsomal protein.

Animals↗
PubMed1996

Permeability of liver microsomal membranes to glucose.

The permeability of rat liver microsomes to glucose has been studied by using (14)C-labelled D-glucose and a light-scattering technique. 1) The microsomal intravesicular apparent isotope space for D-glucose (1mM; after 5 min incubation at 22 degrees C) was 2.34 microl/mg protein, i.e., approximately 72% of the apparent water space. 2) Efflux of [(14)C]D-glucose from microsomal vesicles pre-loaded as in 1) and measured by rapid Millipore filtration after dilution (100 fold) in a glucose-free medium revealed that 15 sec after dilution only 15% of intravesicular glucose was still retained by microsomes. 3) Osmotic behaviour of microsomes upon addition of D-glucose measured by a light-scattering technique revealed a glucose influx, saturable at [D-glucose] > 100 mM, and (partially) inhibited by pentamidine and cytochalasin B. Ascorbic acid, L-glucose and other monosaccharides and related compounds also permeated liver microsomes in a fashion similar to D-glucose. These data indicate the existence of a facilitative transport system(s) for glucose in the membrane of liver endoplasmic reticulum vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1996

Inhibition of rat liver microsomal NADPH cytochrome P450 reductase by glutathione and glutathione disulfide.

Previously we have demonstrated inhibition of lipid peroxidation by reduced glutathione (GSH) in rat liver microsomes that is dependent upon the presence of alpha-tocopheral (alpha-TH) in the membranes. Glutathione disulfide (GSSG) potentiated the inhibitory effect of GSH in an enzymatic (NADPH-dependent) lipid peroxidation system in rat liver microsomes; however, inhibition by GSH + GSSG is independent of alpha-TH. When we repeated these experiments with a non-enzymatic system (ascorbate/ADP) to stimulate lipid peroxidation, GSSG did not potentiate the inhibitory effect of GSH. To delineate the mechanism of inhibition of microsomal lipid peroxidation by GSH + GSSG, we examined the effects of these compounds on cytochrome P-450 reductase (EC 1.6.2.4), an important component of the NADPH-dependent enzymatic lipid peroxidation system. It was observed that GSH alone caused about 25% and 21% inhibition of reductase activity in crude microsomes and partially purified enzyme preparations, respectively. There was no inhibition of reductase activity by GSSG alone in either crude microsomes or partially purified enzyme preparations. However, when added together, GSH and GSSG enhanced the inhibition of reductase activity in crude microsomes and partially purified enzyme by 39% and 56%, respectively. We speculate that one possible mechanism for the inhibition of NADPH-dependent lipid peroxidation by GSH + GSSG is, in part, due to inhibition of NADPH cytochrome P-450 reductase, thus affecting the initiation phase of lipid peroxidation.

Animals↗
PubMed1996

Membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli.

Rat liver microsomal glutathione transferase is a mammalian membrane protein that can be successfully expressed in Escherichia coli in an enzymatically active form. The protein does not form inclusion bodies and is recovered in the membrane fraction. The membrane topology of recombinant rat liver microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. It was shown that lysine-4 of microsomal glutathione transferase is directed towards the outside, whereas lysine-41 faces the inside of the E. coli inner membrane. This shows that microsomal glutathione transferase has an inside-out orientation in E. coli spheroplasts as compared to liver microsomes. This fact enables us to make topology experiments that were previously not possible. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional exposed segments closer to the C-terminus. Thus a polytopic model is suggested for the membrane association of microsomal glutathione transferase.

Animals↗
PubMed1994

Fasciola hepatica: liver microsomal membrane functions in host rat.

Changes in different microsomal membrane functions were measured in the liver of rats 3, 6, or 9 weeks following an oral infection with 20 metacercariae of Fasciola hepatica. The parasitic pathology noted at autopsy was accompanied by increased levels in both plasma aspartate aminotransferase (EC 2.6.1.1) and microsomal gamma-glutamyltransferase (EC 2.3.2.2). Heme oxygenase activity of microsomes was significantly decreased by Weeks 3 and 6 postinfection and this decrease correlates with those of total microsomal cytochrome P450 and certain P450-dependent monooxygenase activities, namely, benzphetamine demethylation, ethoxycoumarin deethylation, and benzopyrene hydroxylation. Microsomal epoxide hydrolase (EC 3.3.2.3) was only altered 6 weeks after the infection. During the early stages of the parasitism, there were decreases in both microsomal calcium uptake and calcium ATPphosphohydrolase activity (EC 2.6.1.1), whereas membrane fluidity, estimated by the order parameter S, was lower in the infected rats than that in the controls. These alterations could be related to the already described increase in liver cytosolic calcium or lipid peroxidation which occurs in experimental fascioliasis.

Animals↗
PubMed1994

Modulation of benzo[a]pyrene bioactivation by glucuronidation in lymphocytes and hepatic microsomes from rats with a hereditary deficiency in bilirubin UDP-glucuronosyltransferase.

UDP-glucuronosyltransferases (UGTs) play a major role in the elimination of nucleophilic metabolites of xenobiotics, such the phenols and quinols of polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BP). In this way, UGTs may prevent the further oxidation of such metabolites to toxic reactive intermediates, which may react with lipids, DNA, RNA, and protein, thereby initiating such toxicities as cellular necrosis, birth defects, and cancer. We have shown previously in vivo and in hepatic microsomes that rats with a hereditary deficiency in bilirubin UGT (Gunn and RHA strains) have decreased glucuronidation of BP metabolites and enhanced BP covalent binding to hepatic DNA and microsomal protein, and enhanced BP embryotoxicity. We further hypothesized that a similar deficiency in BP glucuronidation, with increased BP bioactivation and covalent binding, might be observed in peripheral blood lymphocytes from UGT-deficient rats, which, if true, would suggest the utility of an analogous human lymphocyte model for evaluating the toxicological relevance of human UGT deficiencies. Such an in vitro approach is essential for human studies of environmental chemicals and drugs with a high toxicologic potential. In the current study, [7,10(-14)C]BP was preincubated with NADPH and hepatic microsomes from Wistar rats induced with beta-naphthoflavone. The supernatant from this preincubation, which contained BP reactive intermediates and hydroxylated BP metabolites, was further incubated with uridine diphosphate glucuronic acid and peripheral lymphocytes from heterozygous (j/+) and homozygous (j/j) RHA rats, which are genetically deficient in bilirubin UGT, and from congenic, homozygous (+/+) UGT-normal controls. In vivo, bilirubin glucuronidation was reduced in UGT deficiency, with progressively higher plasma concentrations of unconjugated bilirubin in j/+ and j/j UGT-deficient rats compared to +/+ UGT-normal controls (p < 0.05). In in vitro studies, glucuronide conjugates of BP were measured by high-performance liquid chromatography with a radioisotope detector, and the covalent binding of BP to microsomal protein was measured by liquid scintillation spectrometry. BP glucuronidation and covalent binding, respectively, were decreased and increased to a progressively greater degree in both hepatic microsomes and lymphocytes from j/+ and j/j UGT-deficient rats compared to +/+ UGT-normal controls (p < 0.05). Reduced BP glucuronidation in lymphocytes from UGT-deficient RHA rats correlated with elevated BP covalent binding (R2 = 0.85, p < 0.005). Both BP glucuronidation and BP covalent binding in lymphocytes significantly correlated with those reactions using hepatic microsomes from the same animals. These results in UGT-deficient lymphocytes also accurately reflected the in vivo biotransformation and covalent binding of BP in UGT-deficient rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (Mytilus edulis L.).

The ability of nitrofurantoin (NF) to produce reactive oxygen species (ROS) was investigated in subcellular fractions of digestive gland of the mussel Mytilus edulis in terms of oxygen consumption and the formation of superoxide anion radical (O2-) (measured as SOD-sensitive cytochrome c reduction or SOD-sensitive sensitive .OH production), H2O2 (effect of catalase), and hydroxyl radical (.OH) (iron/EDTA-mediated oxidation of KMBA to ethylene). Additionally, the genotoxic effects of NF were examined using the Salmonella typhimurium umu mutagenicity assay. Microsomal NAD(P)H-dependent oxygen consumption was stimulated by NF, leading to the formation of H2O2. Stimulation of microsomal O2- production by NF was evident for NADH but not NADPH, confirming redox cycling at least with the former coenzyme. No stimulation of O2- production was obvious for cytosolic fraction with either coenzyme. NF stimulated microsomal NAD(P)H-dependent .OH production; the rates of .OH production were greater for NADH than NADPH; and the .OH was indicated to be formed, at least in part, by an iron-catalyzed Haber-Weiss reaction. A role was indicated for a free radical driven Fenton reaction in the NF-stimulated microsomal production of .OH from NADPH. The production of mutagenic species from NF was observed for cytosol but not for microsomes, and the former effects were greater for NADH than NADPH. Overall, the NAD(P)H-dependent microsomal generation of ROS, and the lack of correlation of ROS production with mutagenicity, are considered indicative of the potential of digestive gland to metabolize NF by both one-electron and two-electron reductive pathways. From this and other studies, enhanced ROS production by NF and other redox cycling xenobiotics is indicated to be a widespread phenomenon in aquatic organisms and a potential mechanism of pollutant-mediated toxicity.

Animals↗
PubMed1996

Modulation by iron of hepatic microsomal and nuclear cytochrome P450, and cytosolic glutathione S-transferase and peroxidase in C57BL/10ScSn mice induced with polychlorinated biphenyls (Aroclor 1254).

Exposure of iron-loaded C57BL/10ScSn mice to the polychlorinated biphenyls (PCBs) mixture Aroclor 1254 in the diet (0.01%) for 5 weeks caused massive hepatic porphyria far greater than occurred with PCBs alone. This regime eventually causes hepatocellular carcinoma. Hepatic microsomal ethoxy-, pentoxy-, and benzyloxyresorufin dealkylase activities (respectively EROD, PROD, and BROD) catalyzed primarily by cytochrome P4501A1 and 2B isoenzymes were markedly induced after 2 weeks of diet (when no porphyria had developed) but showed little effect of iron. EROD activity in the nuclear membrane was also induced by the PCBs as was CYP1A1 protein when shown by immunoblotting. Nuclear dealkylase activities of PCBs-treated mice were considerably less than microsomal activities but were stimulated by iron pretreatment. The mechanism of the iron-enhanced toxicity may be due to oxidative damage associated with chronic induction of CYP1A1 isoforms. Lucigenin-enhanced chemiluminescence (CL) by microsomes and nuclear membranes was used as a method to estimate their potential to form reactive oxygen species. Despite CL being induced by PCBs it was less with microsomes from iron-treated mice. In a comparison of a variety of inducers of microsomal cytochrome P450 there was no correlation between inducer, uroporphyrogenic agent, and intensity of CL. On the other hand, cytosolic glutathione S-transferase (GST) activities with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates, were also induced by the PCBs mixture, the induction with DCNB being synergistically potentiated by iron pretreatment. Complementary results were observed by immunocytochemistry using anti alpha-GST antibody. In contrast, total glutathione peroxidase activity and selenium-dependent glutathione peroxidase activity were depressed by PCBs but particularly in mice also administered iron. The results illustrate that PCBs not only induce CYP1A1 in microsomes but also in the nuclear membrane, which may be of significance in the mechanism of the iron-enhanced carcinogenicity of these chemicals. The iron-enhanced induction of GST with accompanying depletion of glutathione peroxidase provides evidence for oxidative processes induced in vivo by the PCBs.

Animals↗
PubMed1997

Cytochrome P450 inactivation during reductive metabolism of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) by phenobarbital- and pyridine-induced rat liver microsomes.

The reductive metabolic activation of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), one of the potential substitutes for the ozone-depleting chlorofluorocarbons and a close structural analogue of the hepatotoxic anesthetic halothane, was investigated in vitro. During incubation of liver microsomes from phenobarbital-(PB) or pyridine-induced (PYR) rats with 0-20 mM HCFC-123 under anaerobic conditions, a dose- and time-dependent depletion of added exogenous glutathione was observed, indicating the formation of reactive metabolites. Under similar incubation conditions, except for the absence of glutathione, 1-chloro-2,2,2-trifluoroethane and 1-chloro-2,2-difluoroethene were detected as products of reductive metabolism of HCFC-123, as previously reported for halothane. As shown previously in our laboratory for halothane, under these conditions HCFC- 123 also caused a statistically significant loss of microsomal cytochrome P450 (P450) as indicated by a decrease of the classical absorption spectrum in the presence of CO. Both glutathione depletion and P450 loss were almost completely prevented by previous saturation of the incubation mixture with CO and were partially prevented by the presence of the free-radical scavenger N-t-butyl-alpha-phenylnitrone or the carbene trapping agent 2,3-dimethyl-2-butene, suggesting that both types of intermediates may be involved. The loss of P450 was associated with a quantitatively similar loss of microsomal heme, as measured by the pyridine hemochromogen reaction, with PB but not with PYR microsomes. Finally, both the P4502E1-specific p-nitrophenol hydroxylase activity in PYR microsomes and the P4502B1/2-specific pentoxyresorufin O-depentylase activity in PB microsomes were significantly inhibited (58 and 53%, respectively) by prior incubation with HCFC-123, suggesting that both isoforms are able to catalyze the activation of this halogenated compound. These results indicate that indeed HCFC-123, like its analogue halothane, is activated reductively to reactive metabolites by at least two P450 isoforms, namely P4502E1 and P4502B1/2. These metabolites, probably free radicals and/or carbene species, may attack the enzyme resulting in modification of the heme group and subsequent loss of catalytic activity.

Animals↗
PubMed1998

Chlorzoxazone metabolism by winter flounder liver microsomes: evidence for existence of a CYP2E1-like isoform in teleosts.

Winter flounder (Pleuronectes americanus) liver microsomes metabolized chlorzoxazone (CZX), a CYP2E1 (cytochrome P450 2E1)- specific substrate, to a single product, 6-OH CZX. The liver microsomal fraction had the highest level of activity, which varied among fish, ranging from 60 to 220 pmol/mg/min. CZX metabolism was linearly related to microsomal protein and dependent on NADPH. The optimal pH range for this activity was from 7.2 to 8.0. Maximal activity was found at 18-22 degreesC (154 +/- 62 pmol/mg/min) but was detectable at all temperatures tested. CZX 6-hydroxylation displayed first-order kinetics as shown by Lineweaver-Burk plots. The average apparent Km, 280 microM, was higher than that reported for human liver microsomes but similar to the Km determined for human CYP2E1 expressed in vaccinia virus. Vmax values ranged from 65 to 141 pmoles/mg/min. The CYP2E1-specific inhibitors, diethyldithiocarbamate (DDC) and aniline, significantly reduced 6-OH CZX production by 80 and 35%, respectively. 7,8-Benzoflavone and ethoxyresorufin, both CYP1A-specific inhibitors, did not significantly inhibit CZX metabolism nor did trioleandomycin (TAO) a CYP3A-specific inhibitor. Diethyldithiocarbamate (DDC), at 50 microM, had no significant effect on winter flounder liver microsomal ethoxyresorufin-O-deethylase activity (EROD), a CYP1A-specific activity. 7,8-Benzoflavone, at 10 microM, inhibited EROD activity by 60%. This is the first report of CYP2E1 activity in teleost liver microsomes.

Aniline Compounds↗
PubMed1999

Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identification of CYP2A6 as a major catalyst.

To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the metabolism of MTBE, ETBE, and TAME.

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

In vitro effects of a smokeless tobacco extract on the production of reactive oxygen species by human oral epidermal cells and rat hepatic mitochondria and microsomes, and peritoneal macrophages.

The possible role of reaction oxygen species in the toxicity of smokeless tobacco was explored. In order to determine possible sources of reactive oxygen species in response to smokeless tobacco, rat peritoneal macrophages (3 x 10(6)/ml) and hepatic mitochondria and microsomes (1 mg protein/ml) from untreated female Sprague-Dawley rats were incubated with an aqueous smokeless tobacco extract (STE) (200 micro g/ml). STE resulted in rapid increases in chemiluminescence with maximum increases occurring at approximately 6 min for the macrophages and 8 min for mitochondria and microsomes. Maximum increases in chemiluminescence of 1.4-, 3.2-, and 3.1-fold relative to control values occurred for macrophages, mitochondria, and microsomes, respectively. Hepatic mitochondria and microsomes (1 mg protein/ml) from female Sprague-Dawley rats were incubated at 37 degrees C for 60 min in the presence of 0-500 micro g/ml STE. Potential tissue damage was measured as lipid peroxidation, and dose-dependent increases of 1.1-2.4-fold occurred in mitochondria and microsomes. Pre-incubation with various oxygen free radical scavengers including superoxide dismutase (SOD) (100 micro g/ml), catalase (100 micro g/ml), SOD + catalase (100 micro g/ml each), mannitol (1.25 mmol/ml), and allopurinol (100 micro g/ml) inhibited STE (200 micro g/ml) induced lipid peroxidation by 15% to 70%. Previous studies in our laboratories strongly suggest that STE induces the production of oxygen free radicals which cause tissue-damaging effects. We therefore examined the cytotoxicity of STE by incubating cultured human oral epidermal carcinoma (KB) cells with STE, and assessing the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular membrane damage. The amount of LDH released by STE was both concentration- and time-dependent. The results demonstrate that oral cells, peritoneal macrophages, and hepatic mitochondria and microsomes produce reactive oxygen species following in vitro incubation with an aqueous extract of smokeless tobacco. Tissue damage in response to STE may occur as the result of reactive oxygen species production.

Animals↗
PubMed1991

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes.

We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, Km values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on Km and Vmax indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID50 values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.

Animals↗
PubMed1991

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes.

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5 microM CaCl2, 5 mM cholate and 100 nM [3H-]Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5) P2) resulted in the formation of [3H-]InsP3. GTP-gamma-S (125 microM) stimulated the production of [3H-]InsP3. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [3H-]PtdIns(4,5)P2 hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca2+. Addition of phorbol ester (100 nM PMA) enhanced the 32P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca2+, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomal GTP-gamma-S-stimulated PtdIns(4,5)P2-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate alpha 1-adrenoceptor mediated PtdIns(4,5)P2 hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.

Animals↗
PubMed1977

Spectral evidence for 2,2,3-trichloro-oxirane formation during microsomal trichloroethylene oxidation.

During aerobic incubation of trichloroethylene with rabbit liver microsomes and NADPH a difference absorption peak appears at 451-452 nm. Trichloroethylene does not form a ligand absorption spectrum with hepatic microsomes reduced by dithionite, or in anaerobic incubates in the presence of NADPH. Addition of trichloroethylene epoxide (2,2,3-trichloro-oxirane) to reduced suspensions of rabbit liver microsomes produces high difference absorption at 452 nm, the optical Ks being approximately 2 mM. Of all possible metabolites of trichloroethylene only trichloroethanol forms absorption in the vicinity of 480 nm, and the broad absorption band reveals relatively low absorption near 450 nm. Dichloroacetyl chloride is the main thermal rearrangement product of trichloroethylene epoxide, and also produces 452 nm absorption in reduced microsomes. However, the difference absorption is 5 times smaller than the absorption produced by the intermediate formed during incubation of trichloroethylene in metabolising liver microsomes. These observations include strong evidence for epoxide formation during microsomal oxidation of trichloroethylene. 14C-labelled trichloroethylene binds irreversibly to hepatic macromolecules in vivo and in vitro. Possible rearrangement pathways of 2,2,3-trichloro-oxirane and reactive intermediates are presented.

Animals↗
PubMed1975

Glucose-6-phosphatase (EC 3.1.3.9) and esterase (EC 3.1.1.1) activities of microsomes prepared from perfused rat livers after partial outflow block or phalloidin poisoning.

Microsomes were prepared from perfused rat livers after different perfusion procedures. The yield of microsomal protein and the kinetic data (Km, Vmax) of glucose-6-phosphatase (3.1.3.9) and esterase (3.1.1.1) activities were analysed in each preparation. No marked differences were detected between conventionally prepared liver microsomes and those from livers perfused 1 hr with an erythrocytes-free medium under the conditions of open outflow. If the outflow pressure was increased artificially, the yield of microsomal protein decreased. The Vmax of both enzymes was markedly increased, whereas the Km values remained unchanged. The same microsomal alterations occurred when perfused rat livers were poisoned with phalloidin in vitro under the condition of open outflow. Our findings indicate that microsomal alterations in livers from poisoned animals might be due to microcirculatory disturbances, and not primary effects of the toxin on the endoplasmatic reticulum.

Animals↗