Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “MICROSOMES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources
PubMed1990

Androgen-dependent renal microsomal cytochrome P-450 responsible for N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene in the BALB/c mouse.

A murine renal microsomal enzyme responsible for the mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) was characterized by its catalytic activity for the mutagenic and metabolic conversion of 3-MeO-AAB. Incubation of 3-MeO-AAB with a renal or hepatic microsome fraction from male BALB/c mice in the presence of NADPH and NADH yielded N-hydroxy and 4'-hydroxy metabolites of 3-MeO-AAB as determined by two-dimensional thin layer chromatography, and the enzyme responsible for the N-hydroxylation was named 3-MeO-AAB N-hydroxylase. A mutagenicity test using Salmonella typhimurium TA98 bacteria as a tester strain has revealed that N-hydroxy-3-MeO-AAB is a potent direct mutagen but that 4'-hydroxy-3-MeO-AAB is not mutagenic. Although 3-MeO-AAB N-hydroxylase activity in liver microsomes showed no sex difference, the enzyme activity in the kidney was detected from male mice but not from females. However, administration of testosterone to female mice induced the enzyme in the kidney. Castration of male mice depressed the activity of 3-MeO-AAB N-hydroxylase in renal microsomes but it little affected the hepatic activity, and on administration of testosterone to the castrated mice the depressed renal microsomal activity recovered to a normal level. The activity of 3-MeO-AAB hydroxylase and the amount of cytochrome P-450 in renal microsomes showed a close correlation. Both renal and hepatic microsomes required NADPH as a main cofactor to mutagenize 3-MeO-AAB and to yield N-hydroxy-3-MeO-AAB from 3-MeO-AAB, and the enzyme activity was strongly inhibited by 7,8-benzoflavone. When the activities of renal and hepatic 3-MeO-AAB N-hydroxylase were compared on the basis of the amount of cytochrome P-450, the renal type enzyme showed about 8 times greater activity than hepatic type enzyme. These results indicate that the kidney contains an androgen-dependent microsomal 3-MeO-AAB hydroxylase which is different from an isozyme present in the liver and which is a new type of cytochrome P-450 isozyme.

Age Factors↗
PubMed1985

Deacetylation to 2-aminofluorene as a major initial reaction in the microsomal metabolism of 2-acetylaminofluorene to mutagenic products in preparations from rabbit lung and liver.

The rabbit pulmonary and hepatic microsomal pathways for the metabolism of 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) to mutagenic products were investigated by means of high performance liquid chromatography and the Salmonella mutagenicity assay. Mutagenic activity approached a maximum with increasing concentrations of AAF incubated with hepatic microsomal preparations and Salmonella; with pulmonary microsomal preparations, mutagenic activity was proportional to the concentration of AAF over the range examined. The mutagenic activities of AF exhibited typical saturation kinetics with both hepatic and pulmonary microsomal preparations. Approximately 7 times more AF than N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) was formed in incubations of AAF (0.5 mM) with hepatic microsomal preparations. When AAF was incubated with pulmonary microsomal preparations, formation of AF, but not N-hydroxy-AAF, was detected. The inclusion of paraoxon in the pulmonary incubations blocked the formation of AF but did not lead to the recovery of any N-hydroxy-AAF. We conclude that the metabolism of AAF to mutagenic products in pulmonary microsomal preparations from rabbits is initiated primarily, if not entirely, by deacetylation of AAF to AF. The mutagenic activity of AAF with the pulmonary microsomal preparations is limited by the deacetylase activity which, like mutagenic activity, exhibits a linear relationship with the concentration of AAF. On the basis of the rates of formation of AF and N-hydroxy-AAF and their mutagenic activities, we estimate that about 60% of the hepatic metabolism of AAF to mutagenic products is dependent upon deacetylation of AAF and subsequent oxidation of the AF formed.

2-Acetylaminofluorene↗
PubMed1980

The in vitro formation of glutathione conjugates with the microsomally activated pulmonary bronchiolar aklylating agent and cytotoxin, 4-ipomeanol.

Using a new high-pressure anion-exchange chromatographic procedure, two radiolabeled conjugates were separated and assayed quantitatively from incubation mixtures containing lung or liver microsomes, NADPH, [3H]5-ipomeanol and glutathione. The same conjugates were obtained also from similar microsomal preparations containing unlabeled 4-ipomeanol, NADPH and [3H]glutathione. The conjugates were not produced in microsomal incubation mixtures containing only [3H]4-ipomeanol and glutathione, or [3H]4-ipomeanol and NADPH, or [3H]glutathione and unlabeled 4-ipomeanol, or in the presence of [3H]glutathione and NADPH alone. Unlike the parent 4-ipomeanol, the conjugates did not have detectable ultraviolet absorbance at 254 nm. Neither of the conjugates arose trough the reduction of 4-ipomeanol to 1,4-ipomeadiol, followed by further metabolism to a metabolite which reacted with glutathione. The total amounts of reactive 4-ipomeanol metabolites generated (estimated by by sum of conjugates plus covalently bound metabolites) were 2 to 3-fold greater in the presence than in the absence of glutathione. Soluble fraction preparations from homogenates of rat lungs or livers did not enhance the corresponding microsome-mediated production of either conjugate, suggesting that glutathione-transferase activities did not play a major role in glutathione conjugate formation with reactive 4-ipomeanol metabolites. The amounts of both conjugates were enhanced greatly in hepatic, but not pulmonary, microsome preparations from animals pretreated with phenobarbital or 3-methylcholanthrene. However, the ratios of the amounts of the two conjugates produced were similar in hepatic and pulmonary microsomal preparations from control rats, or from rats treated with either inducer. These results indicated that the reactive 4-ipomeanol metabolites formed during pulmonary compared to hepatic microsomal metabolism were similar. Moreover, the previously reported enhancement of the hepatic covalent binding and toxicity of 4-ipomeanol caused by pretreatment with 3-methylcholanthrene, but not by phenobarbital, was not likely due to a major change in the kinds or ratios of reactive 4-ipomeanol metabolites produced by hepatic microsomal enzymes after induction with 3-methylcholanthrene compared to phenobarbital.

Alkylating Agents↗
PubMed1993

Alteration in 5'-nucleotidase activities and composition of liver and brain microsomes of developing rats fed different dietary fats.

Four groups of male weanling rats were fed during three months, diets different in the nature of fats and the activity of 5' nucleotidase and fatty acid composition of brain and liver microsomes were studied. Group A were fed a standard commercial diet, group B a fat free-diet and group C and D a fat free-diet, containing respectively 10% of peanut-rapeseed oil and 10% of salmon oil. In brain and liver microsomes, 5'-nucleotidase activity increased throughout the development for all diets (except for the fat-free diet). Slight differences were found in rats fed the peanut-rapeseed oil diet compared to controls estimated at the same time. However, in animals fed the fish-oil diet, 5' nucleotidase had the highest activity in both brain and liver microsomes. Marked changes occurred in the fatty acid patterns of brain and liver microsomes among the various groups. The greatest alterations were found in the liver microsomes. In brain and liver microsomal membranes the fat-free diet induced an increase in monounsaturated fatty acids, an synthesis of eicosatrienoic acid, and a decrease in (n-6) and (n-3) polyunsaturated fatty acids. Animals fed a peanut-rapeseed oil and control diet showed similar fatty acid patterns in liver and brain microsomes. However, when rats were fed a fish-oil diet, the liver microsomal membranes were highly enriched in eicosapentaenoic and docosahexaenoic acids, and simultaneously there was a decrease in arachidonic acid. These results suggest that manipulation of the lipid environment influences 5'-nucleotidase activity by the interaction of the enzyme with specific membrane lipids.

5'-Nucleotidase↗
PubMed2000

Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: role of CYP 2C9 and microsomal glutathione-S-transferase.

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9.

Animals↗
PubMed1988

Ornithine decarboxylase properties: is there a role for a microsome-bound inactivating activity?

Liver microsomes have a strong ornithine decarboxylase (ODC) inactivating capacity in vitro. The present results suggest that this may be involved in regulation of ODC activity in vivo: (1) the ODC inactivating capacity of microsomes appears susceptible to in vivo modulation: a single administration of thioacetamide, which induces ODC, also causes a significant increase in the inactivating capacity of the microsomes; (2) under conditions leading to increased microsome-bound ODC-inactivating capacity (e.g. liver from thioacetamide-treated rates versus regenerating liver) ODC displays a greater thermal lability and inactivability in vitro. A possible involvement of this microsomal activity in an autoregulatory pathway of ODC is suggested by the fact that it is induced by the administration of polyamines. However, inhibition of ODC activity by alpha-difluoromethylornithine does not prevent the increase of the microsomal activity caused by thioacetamide. Thus, polyamine biosynthesis does not appear to be an absolute requirement for induction of the microsomal ODC-inactivating capacity. The apparent half-life of ODC in vivo, as evaluated after cycloheximide administration, does not appear to correlate with the microsomal ODC-inactivating capacity content and the stability properties of ODC in vitro.

Animals↗
PubMed1990

Metabolism of halazepam by rat liver microsomes: stereoselective formation and N-dealkylation of 3-hydroxyhalazepam.

Metabolism of halazepam [7-chloro-1,3-dihydro-5-phenyl-1-(2,2,2-trifluoroethyl)-2H-1,4-benzod iazepin- 2-one, HZ] was studied by incubation with liver microsomes prepared from untreated, phenobarbital (PB)-treated, and 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats. Metabolites of HZ were separated by normal-phase HPLC. Relative rates of HZ metabolism by liver microsomes prepared from untreated and treated rats were PB-treated much greater than untreated greater than 3MC-treated at low concentration of microsomal enzymes (0.25 mg protein per ml of incubation mixture) and PB-treated much greater than 3MC-treated approximately untreated at high concentration of microsomal enzymes (2 mg protein per ml of incubation mixture). The relative amounts of major metabolites were found to be 3-hydroxy-HZ (3-OH-HZ) greater than N-desalkylhalazepam (NDZ, also known as N-desmethyldiazepam and nordiazepam) much greater than oxazepam (OX) for all three rat liver microsomal preparations and the distribution of metabolites was independent of microsomal enzyme concentrations. Enantiomers of 3-OH-HZ were resolved by HPLC on a Chiralcel OC column (cellulose trisphenylcarbamate coated on silica gel, particle size 10 microns). 3-OH-HZ enantiomeres have racemization half-lives of approximately 150 min in pH 4, 7.5, and 10 aqueous solutions. 3-OH-HZ formed in the metabolism of HZ by liver microsomes prepared from untreated and treated rats were found to have 3R/3S enantiomer ratios of 37/63 (untreated), 55/45 (PB-treated), and 36/64 (3MC-treated), respectively. N-dealkylation of 3-OH-HZ by liver microsomes from PB-treated rats was substrate enantioselective; the 3R-enantiomer was N-dealkylated faster than 3S-enantiomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Anti-liver-kidney microsome antibody is a marker for the rat hepatocyte endoplasmic reticulum.

Human sera, containing anti-liver-kidney microsome antibody as demonstrated by indirect immunofluorescence, were obtained from a subgroup of young patients with autoimmune chronic hepatitis. The anti-liver-kidney microsome antibody-positive sera were used to study the localization of the liver-kidney microsome antigen in hepatocytes. Immunoblot analysis of microsomal subfractions, lysosomal membranes, plasma membranes, mitochondria and purified ribosomes obtained from rat liver demonstrated that this antibody recognizes a protein of 50 kD present only in endoplasmic reticulum membranes. Immunogold labeling of ultrathin frozen sections and immunoperoxidase staining of 11 to 15 micron cryostat sections were used to detect the liver-kidney microsome antigen in rat liver tissue. The anti-liver-kidney microsome antibody binds to antigenic domains on the cytoplasmic face of smooth and rough endoplasmic reticulum membranes of hepatocytes. No labeling was observed of the Golgi apparatus, peroxisomes, mitochondria, lysosomes, nuclei or plasma membranes. Not only was the antigen recognized by the anti-liver-kidney microsome antibody specific for endoplasmic reticulum membranes, but it was also specific for the endoplasmic reticulum of hepatocytes only, since no labeling was observed in any organelle of Kupffer or endothelial cells. Therefore, the anti-liver-kidney microsome antibody can be considered as a marker for endoplasmic reticulum in rat hepatocytes.

Antigens↗
PubMed1987

Inhibition of hepatic microsomal lipid peroxidation by endogenous glycogen in the rat.

The effect of endogenous glycogen on lipid peroxidation was examined in hepatic microsomes from rats. Microsomes were prepared to retain endogenous hepatic glycogen (Pg+) or to minimize it (Pg-). The indices of lipid peroxidation examined included the rate of NADPH-dependent formation of malondialdehyde (MDA) and the concomitant destruction of cytochrome P-450 and decline in the linearity of benzphetamine N-demethylase activity in microsomes. Cytochrome P-450 was destroyed during benzphetamine N-demethylation in microsomes with the loss being more extensive in Pg- than in Pg+. The destruction of cytochrome P-450 and the concomitant loss in linearity of benzphetamine N-demethylation in Pg- were prevented by added EDTA. Added linoleic acid hydroperoxide (LAHP) also caused a time-dependent loss of cytochrome P-450 in microsomes with the rate being greater in Pg- than in Pg+. The results show that glycogen inhibits hepatic microsomal lipid peroxidation and suggest that variations in glycogen content may contribute to disparities in in vitro oxidative activities between different microsomal samples. Such disparities may be minimized by the removal of glycogen during the preparation of microsomes and then supplementing the incubation mixtures with EDTA. The in vivo relevance of the observed antioxidant effect of glycogen is discussed in terms of the possible modulation by the polysaccharide of hepatotoxicity by agents whose effects may be mediated by lipid peroxidation.

Animals↗
PubMed1978

Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding.

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and "rough-inverted" vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them.

Animals↗
PubMed1993

ESR studies on the production of reactive oxygen intermediates by rat liver microsomes in the presence of NADPH or NADH.

Experiments were carried out using spin-trapping ESR spectroscopy to evaluate in a quantitative and kinetic manner the production of reactive oxygen intermediates by rat liver microsomes. Comparisons between the effectiveness of NADH and that of NADPH in catalyzing microsomal generation of reactive oxygen intermediates were made. Superoxide production was determined by assaying the generation of superoxide dismutase-sensitive stable nitroxyl radicals formed from 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine. Identical spectra were produced when microsomes were incubated with NADH or NADPH; reaction rates were linear for at least 10 min of reaction and were about three- to fourfold greater with NADPH. In the presence of iron, microsomes catalyze the production of hydroxyl radical during electron transfer. Initial experiments utilizing 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) as the spin-trapping agent proved unsatisfactory for the microsomal system as adequate kinetics could not be determined in view of the rapid decay of the DMPO-OH adduct, as well as secondary adducts such as DMPO-CH3 or DMPO-hydroxyethyl (HER). The spin-trapping agent alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN) was evaluated. POBN-OH adducts were somewhat more stable but also rapidly decayed after 2 to 3 min. However, production of POBN-CH3 and POBN-HER adducts was proportional to that of microsomal protein, increased with time over a 5- to 15-min period, and then was relatively stable. The redox cycling agent paraquat increased POBN-HER adduct formation twofold. Formation of the adduct with either NADH or NADPH required an iron catalyst and did not occur in the presence of the iron chelator desferrioxamine. Ferric EDTA was most reactive in catalyzing production of the adduct, ferric DTPA was 60 to 70% as effective, and ferric ATP was 20 to 30% as effective as ferric EDTA, with both reductants. Irrespective of the iron complex, rates of POBN-HER formation with NADH were about 70% those of NADPH. Formation of the adduct was inhibited by catalase, mannitol, and GSH, but not superoxide dismutase. These experiments support the usefulness of POBN plus ethanol for kinetic studies on the production of .OH by microsomes and validate that microsomes in the presence of an iron catalyst generate .OH not only with NADPH as cofactor, but also with NADH.

Animals↗
PubMed1993

Oxygen consumption and oxyradical production from microsomal reduction of aqueous extracts of cigarette tar.

Aqueous extracts of cigarette tar (ACT) contain a quinone-hydroquinone-semiquinone system that undergoes autoxidation to produce superoxide and hydrogen peroxide. In this paper we ask whether the production of activated oxygen species from this quinone system can be enhanced via an NAD(P)H-dependent autoxidation in which electrons are donated from electron transport components of the microsomal system to electron acceptors in ACT such as quinones and/or metal chelates. Therefore, the ability of ACT to enhance the oxidation of NADPH, consumption of oxygen, and production of oxyradicals during microsomal electron transport has been investigated. Rates of NADPH oxidation and oxygen consumption are enhanced in a concentration-dependent manner by both aqueous and dimethyl sulfoxide (DMSO) extracts of tar over that observed in the presence of microsomes alone. Over the time course studied, neither NADPH oxidation nor oxygen consumption by tar extracts is observed in the absence of microsomes. Microsomal NADPH oxidation is enhanced by 1,6- and 1,3-benzo[a]pyrene quinones and by iron-EDTA; per unit weight, the enhancement by these compounds is similar to that of tar. NADPH oxidation in the presence of tar and absence of iron-EDTA is inhibited by about 15% by the addition of deferoxamine to the reaction mixture. Chrysene quinone enhances NADPH oxidation to a much greater extent than does either of the benzo[a]pyrene quinones or tar. Inhibition of tar-stimulated microsomal oxygen uptake by superoxide dismutase, stimulation of oxygen uptake by azide (an inhibitor of catalase), and the presence of spin-trappable superoxide anion radical all indicate the reduction of tar components to superoxide and hydrogen peroxide. The similar effects of tar on NADPH oxidation catalyzed either by microsomes or by purified NADPH-cytochrome P450 reductase suggest that cytochrome P450 reductase is an important locus of electron transfer from microsomes to components of cigarette tar extracts. These data suggest that the cytotoxic and mutagenic effects of smoking may be mediated by metabolic activation of quinones and, to a lesser extent, metal chelates in cigarette tar to oxyradicals.

Animals↗
PubMed1994

Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli.

Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.

Alkylation↗
PubMed2000

Evidence for requirement of NADPH-cytochrome P450 oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system.

Rabbit antibodies raised against the hydrophilic part of microsomal NADPH-cytochrome P450 oxidoreductase (denoted fpT) demonstrated a marked ability to inhibit NADPH-sterol Delta7-reductase activity. In addition, trypsin and proteinase K treatment of microsomes removed almost all microsomal electron transfer constituents from the microsomes, but the Delta7-reductase activity could be reconstituted by adding detergent-solubilized NADPH-cytochrome P450 oxidoreductase (denoted OR). Furthermore, after solubilization from microsomes, the Delta7-reductase activity could be reconstituted with OR in a DEAE-cellulose column chromatography eluate fraction, which contained little OR activity. In the microsomal system, carbon monoxide, ketoconazole, and miconazole, specific inhibitors of cytochrome P450, had no effect on Delta7-reductase activity. These results provide the first evidence of an essential requirement of OR, which is distinct from cytochrome P450, in the NADPH-sterol Delta7-reductase system. EDTA, o-phenanthroline and KCN markedly lowered Delta7-reductase activity in a dose-dependent manner. Among metal ions tested, only ferric ion restored the reductase activity in the EDTA-treated microsomes. These results sugguest that NADPH-sterol Delta7-reductase is membrane-bound iron-dependent protein embedded in the microsomal lipid bilayer.

Animals↗
PubMed1996

In vitro effect of lead on Ca(2+)-ATPase in synaptic plasma membranes and microsomes of rat cerebral cortex and cerebellum.

Earlier investigations from this laboratory suggest that lead interferes with the calcium homeostasis of rat brain through modulation of an inositol polyphosphate second-messenger system. The present investigation was initiated to study the comparative effects of lead chloride and lead acetate on synaptosomal and microsomal Ca(2+)-ATPase of rat cerebral cortex and cerebellum in vitro. The synaptic plasma membranes and microsomes were prepared by using sucrose gradient (1.2-0.8 M). The assay of Ca(2+)-ATPase was done by hydrolysis of ATP and the liberated inorganic phosphate was estimated. Both lead chloride and lead acetate at micromolar concentrations significantly inhibited the Ca(2+)-ATPase of synaptic plasma membranes and microsomes of cerebral cortex and cerebellum in a concentration-dependent manner. The IC50 values of Ca(2+)-ATPase for both lead salts in synaptosomes were significantly lower (P < 0.05) than that of microsomes, indicating more sensitivity. Significantly (P < 0.05) lower IC50 values for both synaptosomal and microsomal Ca(2+)-ATPase were obtained for lead acetate than for lead chloride. The results suggest that lead acetate is more potent than lead chloride in inhibiting the Ca(2+)-ATPase. The microsomal Ca2+ uptake was also studied in cerebellum and cerebral cortex in the presence of different concentrations of both the lead salts. However, these lead salts in vitro did not reveal a significant (P < 0.05) change in the microsomal Ca2+ uptake of cerebellum and cerebral cortex. But earlier investigations indicated that in vitro lead (0.25-2 microM) inhibits inositol 1,4,5-triphosphate-mediated Ca2+ uptake and release in microsomes of rat cerebellum.

Adenosine Triphosphate↗
PubMed1993

Contrasting effects of 1,1,1-trichloroethane on [14C]vinyl chloride metabolism and activation in hepatic microsomes from phenobarbital- and isoniazid-treated rats.

The interaction of 1,1,1-trichloroethane (TCE), a widely used chlorocarbon solvent, on the metabolism and activation of [14C]-vinyl chloride in rat hepatic microsomes was investigated to determine the effects of combined exposures to these compounds. In microsomes from phenobarbital (PB)-treated rats, TCE increased vinyl chloride-protein binding and vinyl chloride aqueous metabolite formation over twofold when vinyl chloride 0.32% (v/v) and TCE (0.65%) are incubated together. In contrast, under similar incubation conditions, TCE inhibited vinyl chloride metabolism and protein binding up to 45% in microsomes from isoniazid-treated animals. TCE also inhibited vinyl chloride metabolism and binding in microsomes from untreated rats, but to a lesser degree. Like the effect of TCE on vinyl chloride-protein binding, TCE increased vinyl chloride binding to DNA approximately 130% in microsomes from PB-treated rats, yet inhibited vinyl chloride-DNA binding in microsomes from isoniazid-treated and untreated animals. Consistent with TCE effects on vinyl chloride binding and aqueous metabolite production, TCE further increased cytochrome P450 loss due to vinyl chloride metabolism in microsomes from PB-treated rats, but was inhibitory to cytochrome P450 loss due to vinyl chloride metabolism in microsomes from isoniazid-treated and untreated rats. These data demonstrate that the relatively metabolically inert solvent, 1,1,1-trichloroethane, can directly increase vinyl chloride metabolism and activation catalyzed by the phenobarbital-inducible isozymes, but is inhibitory toward vinyl chloride metabolism catalyzed by the isoniazid-inducible CYP2E1.

Animals↗
PubMed1990

Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450.

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.

Acetates↗
PubMed1982

Identity of microsomal glutathione S-transferases.

Mouse liver microsomes were prepared by repeated washing, homogenization, and centrifugation until almost no more soluble enzymes were found in the supernatant of the last centrifugation. About 0.09% of the total glutathione S-transferase activity and comparable amount of soluble enzymes were detected in microsomes solubilized with Emulgen 913. By double immunodiffusion, microsomal glutathione S-transferase were shown to have a complete immunological identity with cytosolic F2 and F3 transferase from mouse liver. By Sephadex gel filtration chromatography in 1% Emulgen 913, part of the microsomal transferase activity (20 to 50%) was shown to be associated with the microsomal membrane protein fraction and appeared in the void volume. Partially purified microsomal transferases were found to have molecular weights, isoelectric points and Km's for substrate and GSH which are comparable to those of soluble liver transferases. This study seems to suggest that the presence of glutathione S-transferases in microsomes is the result of specific and nonspecific association between the microsomal membrane and soluble liver transferases.

Animals↗