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PubMed1984

The interaction of amiloride analogues with the Na+/H+ exchanger in kidney medulla microsomes.

The effects of ten amiloride analogues on Na+-H+ exchange in rabbit kidney medulla microsomes have been examined. Most of the analogues appeared to inhibit Na+ uptake into the microsomes more effectively than did amiloride either in the presence or absence of a pH gradient. However, the analogues were also capable of stimulating Na+ efflux from the microsomes at concentrations somewhat higher than the concentrations at which they inhibited Na+ influx. The concentrations at which the analogues stimulated Na+ efflux were about 2-4-times higher than the concentrations at which they blocked influx. This suggested that the two processes were related. The analogues that stimulated efflux most effectively (the 5-N-benzyl-amino analogue of amiloride and the 5-N-butyl-N-methylamino analogue) were shown to induce completely reversible effects. These analogues did not stimulate L-[3H]glucose efflux from medulla microsomes which ruled out nonspecific vesicle destruction or reversible detergent effects. These analogues also induced Na+ efflux from microsomes in the presence of high concentrations of added buffer, which ruled out weak-base uncoupling effects. The possibility exists that these analogues are carried into the microsomes via the Na+-H+ exchange protein and that this permits them to both block Na+ influx into the microsomes and stimulate Na+ efflux as well.

Amiloride↗
PubMed1988

Protein-mediated fusion of liposomes with microsomal membranes of Aspergillus niger: evidence for a complex mechanism dealing with membranous and cytosolic fusogenic proteins.

Membrane fusion is a fundamental and wide-spread phenomenon in the functioning of cells. Many studies were carried out concerning fusion of plasma membranes as for example cell-cell fusions or uptake by cells of lipid-enveloped viruses. The present study deals with the interaction of intracellular membranes of Aspergillus niger with artificial membranes (liposomes). Association is monitored by the uptake of radioactive liposomes by fungal microsomal membranes. The discrimination between aggregation and pure fusion is done by layering the liposomes-microsomes mixture on a continuous sucrose gradient. The accurate quantitation of the fusion phenomenon is monitored with a fluorescent assay based on resonance energy transfer (Struck, D.K. et al. (1981) Biochemistry 20, 4093-4099). Both methods show that, at physiological pH, there is a spontaneous fusion of microsomes with cholesterol-free liposomes. This phenomenon is protein dependent as trypsinized microsomal membranes are no longer able to fuse with liposomes. Biological significance of the fusion process has been demonstrated using microsomal intrinsic protein mannosylation assay; the enhancement of the lipid to protein ratio due to the fusion of liposomes with microsomes of A. niger results in an increase in the rate of endogenous proteins mannosylation. Moreover, cytosolic proteins of A. niger promote the fusion of any kind of liposomes with microsomes.

Aspergillus niger↗
PubMed1988

Characteristics of GTP-mediated microsomal Ca2+ release.

Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.

Animals↗
PubMed1981

Topography of glycerolipid synthetic enzymes. Synthesis of phosphatidylserine, phosphatidylinositol and glycerolipid intermediates occurs on the cytoplasmic surface of rat liver microsomal vesicles.

The topography of glycerolipid biosynthetic enzymes within the transverse plane of rat liver microsomal vesicles was investigated: (1) by use of the impermeant inhibitor, mercury-dextran; (2) by use of proteases; and (3) by determining whether the enzyme activities are latent. The seven enzyme activities investigated (dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate oxidoreductase, phosphatidic acid : CTPcytidyltransferase, CDPdiacylglycerol : inositol phosphatidyltransferase, 2-monoacylglycerol acyltransferase, diacylglycerol kinase, and the serine base exchange enzyme) function in phosphatidylinositol and phosphatidylserine synthesis and at intermediate levels in glycerolipid synthesis including steps of ether lipid synthesis. Mercury-dextran inhibited four of these enzymes greater than 60% in intact microsomal vesicles. One or more of the proteases employed (chymotrypsin, trypsin and pronase) inactivated each of the seven enzyme activities in intact microsomal vesicles. These two approaches indicate that each of these enzymes has important domains located on the cytoplasmic surface of microsomal vesicles. These enzyme activities could be assayed in intact microsomal vesicles. None appeared to be highly latent, indicating that substrates have free access to active sites. One substrate for each of these enzymes had been shown previously to be unable to cross the microsomal membrane. These data indicate that the active sites of these enzymes are located on the cytoplasmic surface of microsomal vesicles. It is concluded that the synthesis of phosphatidylserine and phosphatidylinositol, intermediates of ether lipid formation and other intermediates of glycerolipid synthesis occur asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. These findings and our previous investigations on the topography of seven enzymes of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine biosynthesis (Ballas, L.M. and Bell, R.M., Biochim. Biophys. Acta 602, (1980) 578-590) indicate that the synthesis of the major cellular glycerolipids occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum.

Acyltransferases↗
PubMed1984

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed beta-carotene and 13-cis-retinoic acid.

The fatty acid composition of microsomal lipids and the activities of delta 9- and delta 6-desaturases in liver microsomes of rats fed diets supplemented with beta-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg beta-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for delta 9-desaturase and delta 6-desaturase activities. The activity of delta 9-desaturase was lower in liver microsomes of rats fed beta-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal delta 6-desaturase activity was, however, higher in liver of rats fed 13-cis retinoic acid; there was no effect of beta-carotene on delta 6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding beta-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.

Animals↗
PubMed1986

Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa.

This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.

Alcohol Oxidoreductases↗
PubMed1990

Phosphatidylinositol synthetase activities in neuronal nuclei and microsomal fractions isolated from immature rabbit cerebral cortex.

The synthesis of phosphatidylinositol was studied using a nuclear fraction N1, a microsomal fraction P3, rough (R) and smooth (S) microsomal fractions and a microsomal fraction P derived from isolated nerve cell bodies. Each fraction was prepared using cerebral cortices of 15-day-old rabbits. In assays using CDP-diacylglycerol (prepared from egg phosphatidylcholine) and myo[3H]inositol at pH 7.4, fraction N1 had the highest maximal specific rates of phosphatidylinositol synthetase (EC 2.7.8.11) (expressed per mumol phospholipid in the fraction). However the three microsomal fractions achieved maximal specific activities at liponucleotide concentrations close to 50 microM, while fraction N1 required 200 microM concentrations. In certain cases (25-120 microM CDP-diacylglycerol, and at higher pH values) fraction R had specific activities which equalled or surpassed those of N1. However, with respect to inositol, fraction N1 had a distinctly lower Km than was shown for fractions R or P3. Each of the microsomal fractions and N1 required Mg2+ for the reaction, but for N1, maximal rates could be sustained at 0.1 mM, while for the microsomal fractions the optimal Mg2+ concentration was 1 mM. For each fraction Mn2+ could not replace Mg2+ in the reaction and Mn2+ was inhibitory. The optimal pH for the reaction was between 8.0 and 9.0. Phosphatidylinositol synthetase could also be shown using fraction N1 enriched in endogenous CDP-diacylglycerol. The relatively high specific activities of fraction N1, and the differences found between N1 and the microsomal fractions, for optimal CDP-diacylglycerol and Mg2+ concentrations and for Km values for inositol, support the existence of a neuronal nuclear phosphatidylinositol synthetase.

Animals↗
PubMed1991

Evidence against cytochrome b5 involvement in liver microsomal fatty acid elongation.

This study provides strong evidence against cytochrome b5 participation in the first reduction step-beta-ketoreduction-of rat liver microsomal fatty acid chain elongation. Several lines of evidence led to this conclusion: (a) beta-ketoreductase was not inducible by diet conditions since its activity was the same in microsomes from fasted rats and in rats fed a fat-free diet. Consequently, its activity was appreciable in microsomes from fasted rats. Nevertheless, cytochrome b5 reoxidation rate was not stimulated by adding beta-ketopalmitoyl-CoA to the latter microsomes. This suggests that it is not the activated beta-ketoreductase which stimulates the cytochrome b5 reoxidation rate, but another electron acceptor. (b) The delta 9-desaturase, present in microsomes from rats fed a fat-free diet, was totally inhibited by 4 mM KCN; beta-ketopalmitoyl-CoA or malonyl-CoA stimulated the reoxidation rate of cytochrome b5 but this increase was also inhibited by 4 mM KCN. This suggests that delta 9-desaturase is involved in the stimulation and shows that any inhibitor of delta 9-desaturase, including cytochrome b5 antibodies, may induce elongation inhibition. (c) NADH-dependent beta-ketoreductase activity was partially purified from Triton X-100 solubilised microsomes, in a fraction essentially free of cytochrome b5. Furthermore, when the fraction containing cytochrome b5 and NADH-cytochrome-b5 reductase was added to the fraction containing beta-ketoreductase activity, no increase in beta-ketoreductase activity was observed. Stearoyl-CoA desaturase activity which is also present in microsomes from rats fed a fat-free diet led to the results which have been misinterpreted in the conclusions of previous studies.

Alcohol Oxidoreductases↗
PubMed1989

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions.

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.

Animals↗
PubMed1983

Effects of cyclic AMP and protein kinase on calcium uptake in a microsomal fraction from guinea pig taenia caecum.

A microsomal fraction was isolated from guinea pig taenia caecum by differential centrifugation. Activities of ouabain-sensitive (Na+, K+)-ATPase, 5'-nucleotidase and NADPH-cytochrome c reductase were enriched in the microsomal fraction. On the other hand, less cytochrome c oxidase and monoamine oxidase were contained in this fraction. These results suggest that the microsomal fraction used in this study was derived from both sarcolemma and sarcoplasmic reticulum. Ca2+ uptake by this fraction was strictly dependent on the presence of ATP and was facilitated by oxalate. An ATP-regenerating system was required for the determination of Ca2+ uptake, when a lower concentration of ATP (e.g. 0.25 mM) was used. Phosphorylation of the microsomal fraction was doubled when these membranes were incubated in the presence of cyclic AMP plus cyclic AMP-dependent protein kinase (protein kinase). When the microsomal fraction was pretreated with cyclic AMP plus protein kinase, Ca2+ uptake was stimulated. The increases in microsomal phosphorylation and Ca2+ uptake were significantly correlated (P less than 0.01). This stimulation of Ca2+ uptake by microsomal phosphorylation was observed only in the presence of protein kinase, oxalate, and low Ca2+ and Mg2+ concentrations. The results suggest that stimulation of Ca2+ uptake may be the mechanism by which cyclic AMP is involved in beta-adrenergic relaxation of smooth muscle.

Adenosine Triphosphate↗
PubMed1983

Changes in some rat hepatic microsomal components induced by prolonged administration of chloroquine.

Alterations in microsomal drug metabolizing enzymes and phospholipids following prolonged exposure to chloroquine have been investigated. The levels of microsomal aminopyrine-N-demethylase, aniline hydroxylase and both microsomal and cytosolic glutathione-S-transferase are reduced in treated rats. Microsomal epoxide hydrolase is unaffected by the treatment. An increase in the cholesterol-phospholipid ratio and a decrease in the phosphocholine-sphingomyelin ratio occur. There is a general reduction in the total microsomal phospholipid level though the percentage content of sphingomyelin is higher in all cases. The reduction in microsomal phospholipid level is probably due to a reduced incorporation of acetate into microsomal phospholipids as shown in this study using radioactive sodium acetate.

Aminopyrine N-Demethylase↗
PubMed1983

Modifications of carcinogen metabolism in hepatic microsomes of suckling young by 3-methylcholanthrene or beta-naphthoflavone administered to lactating rats.

The effects of treating lactating rats with 3-methylcholanthrene (3-MC) or beta-naphthoflavone (beta-NF) (three i.p. injections of 20 or 40 mg compound/kg of body weight) on hepatic microsomal enzymes of their suckling young were examined. This treatment had no apparent effect on the contents of cytochromes P-450 and b5 or on the activities of NADH- and NADPH-cytochrome c reductases in hepatic microsomes of the pups. However, these microsomes had 8- and 6-fold increased capacities for hydroxylations of benzo[a]pyrene (B[a]P) and N-2-fluorenylacetamide (2-FAA) respectively. These increases were about 5-fold greater in the hepatic microsomes of the dams, in which they were inhibited by 0.1 mM alpha-naphthoflavone (alpha-NF) in vitro 72-81 and 89-95% and by 0.1 mM beta-NF in vitro 12-41 and 60-76% respectively. In the pups, the induced activities were also inhibited, whereas the basal hydroxylations of B[a]P and 2-FAA were stimulated by alpha-NF 2.7- and 5.0-fold and by beta-NF 1.4- and 2.4-fold respectively. The inhibition of the induced hydroxylations by alpha-NF and beta-NF may be explained by their higher affinities (Ks, 0.14 and 0.28 microM, respectively) than those of B[a]P and 2-FAA (Ks, 4.4 to 8.8 and 2.4 to 3.1 microM, respectively) for cytochrome P-450. Whereas beta-NF gave a type I binding spectrum, alpha-NF gave a spectrum composed of type I and reverse-type I elements. Analysis of metabolites of 2-FAA showed differences in their type and amounts formed by hepatic microsomes of beta-NF-treated lactating rats and their pups. Thus, in the dams the formation of 1-, 3-, 5-, 7-, 9- and N-hydroxy-2-FAA was increased by 9-, 30-, 40-, 5-, 20- and 5-fold respectively. In the pups, the formation of 1-, 3-, 5-, 7- and N-hydroxy-2-FAA was increased by 2-, 30-, 18-, 4- and 27-fold respectively. All these increased hydroxylations were inhibited by 0.1 mM alpha-NF in vitro. In the hepatic microsomes of pups from the corn oil-treated dams, alpha-NF stimulated all ring-hydroxylations, but not N-hydroxylation of 2-FAA. The results support earlier findings that microsomal enzymes differ in immature and mature rat liver and suggest that N-hydroxylation of 2-FAA, the activation required for carcinogenesis, and specific ring-hydroxylations are catalyzed by different cytochrome P-450 isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Acetylaminofluorene↗
PubMed1983

Effect of hydrogen peroxide on the initiation of microsomal lipid peroxidation.

Hydrogen peroxide reacts with reduced transition metals to generate the highly reactive hydroxyl radical (X OH), most often proposed as the predominant species for initiating microsomal lipid peroxidation. To assess the potential involvement of X OH, generated from hydrogen peroxide, in microsomal lipid peroxidation, we have altered the concentration of microsomal hydrogen peroxide and measured the resulting rates of malondialdehyde production. Hydrogen peroxide concentration in microsomes was changed by adding exogenous catalase, by washing to reduce both endogenous catalase activity and hydrogen peroxide-dependent glutathione oxidase activity, and by inhibiting endogenous catalase activity with azide in either the presence or absence of exogenous hydrogen peroxide. In only one instance was the rate of lipid peroxidation affected; exogenous hydrogen peroxide added to microsomes, previously incubated with azide, inhibited lipid peroxidation, the opposite effect from that predicted if X OH, generated from hydrogen peroxide, is actually the major initiating species. Neither these results, nor the inability of known X OH traps to inhibit microsomal lipid peroxidation, support the role of free hydrogen peroxide in the initiation of microsomal lipid peroxidation.

Animals↗
PubMed1984

Metabolism-dependent binding of the chlorinated insecticide DDT and its metabolite, DDD, to microsomal protein and lipids.

Dichlorodi[U-14C]phenyltrichloroethane ( [14C]DDT), incubated with rat hepatic microsomes and NADPH, produced reactive intermediates which covalently bound to microsomal protein and lipids. In atmospheric oxygen, DDT bound to microsomal protein; however, binding was increased up to approximately 70% by oxygen depletion. Low levels of [14C]DDT binding to microsomal lipids occurred under atmospheric oxygen but, in contrast to protein binding, DDT-phospholipid binding was increased up to 20-fold by oxygen depletion. Dichlorodiphenyldichloroethane (DDD) was rapidly formed from DDT under anaerobic conditions, although when DDD was utilized as substrate, binding to microsomal protein occurred only in the presence of oxygen. Sodium dithionite, added to microsomes, produced [14C]DDT phospholipid and protein binding, and DDD formation, but failed to support DDD metabolism or binding. The data are consistent with the reductive formation of a DDT free-radical intermediate that led to the formation of DDD and that was bound preferentially to microsomal lipids.

Animals↗
PubMed1985

Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.

Cytochrome b5 has recently been shown to be required in the reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane [E. Canova-Davis and L. A. Waskell, J. biol. Chem. 259, 2541 (1984)]. To determine whether this observation in the reconstituted system was merely dependent on the particular ratios of the various components or some other fortuitous, unknown factor, or whether cytochrome b5 plays a role in the liver microsomal metabolism of methoxyflurane, the following studies were undertaken. Antibody to rabbit holocytochrome b5 was raised in guinea pigs. The antibody to cytochrome b5 was able to inhibit 75% of the microsomal metabolism of methoxyflurane. This same antibody also inhibited methoxyflurane metabolism in the reconstituted system. When the antibody to cytochrome b5 was treated with purified cytochrome b5 before addition to the microsomes, it did not inhibit methoxyflurane metabolism. Furthermore, the antibody to cytochrome b5 did not inhibit the microsomal metabolism of benzphetamine. This suggests that cytochrome b5 was required for the microsomal metabolism of methoxyflurane. It is possible that cytochrome b5 functioned in the metabolism of methoxyflurane by retaining a specific conformation of cytochrome P-450 and not by transferring the second electron to cytochrome P-450. To explore this possibility, cytochrome b5 was reconstituted with Mn3+-protoporphyrin IX. The Mn3+-protoporphyrin IX derivative retained the conformation of cytochrome b5 but not its electron transfer properties. This manganese derivative of cytochrome b5 was unable to stimulate the metabolism of methoxyflurane. The study demonstrated that cytochrome b5 was obligatory for the microsomal metabolism of methoxyflurane, whereas it was not required for the microsomal N-demethylation of benzphetamine. Moreover, the heme moiety of cytochrome b5 functioned to transfer electrons in this reaction.

Animals↗
PubMed1985

Microsomal calcium-accumulating ability of bovine coronary artery and aorta.

Calcium uptake and binding activities of microsomal fractions from bovine coronary artery and aorta were examined. The isolated microsomal fraction of the coronary artery and aorta showed 7- to 8-fold higher glucose-6-phosphatase activity and 4- to 6-fold higher NADPH-cytochrome c reductase activity as compared with the corresponding values for the homogenate fraction. Coronary artery and aorta microsomal calcium uptake activities were 118 and 159 nmoles Ca2+/mg protein/10 min in the presence of 100 microM CaCl2, respectively. These activities for bovine vascular smooth muscle microsomes are higher than those of other species investigated. The calcium uptake activities were dependent on calcium concentrations ranging from 5 to 50 microM in the assay medium. The onset of the reaction for aorta microsomal calcium uptake was faster than that for the coronary artery. The calcium uptake activity was also dependent on ATP, but it was practically independent of oxalate ions in the assay medium. Microsomal calcium binding activities of the coronary artery and aorta were maximal at 20 min of incubation under the present experimental conditions. A lower Km value of the aortic calcium binding for ATP was obtained as compared with that for the coronary artery. The present experiment explored several characteristics of the microsomal calcium-accumulating ability of vascular smooth muscle, which provides meaningful information for further study on cellular calcium movements in vascular smooth muscle.

Adenosine Triphosphatases↗
PubMed1987

Characterization of the cytochrome P-450 monooxygenase system of hamster liver microsomes. Effects of prior treatment with ethanol and other xenobiotics.

The cytochrome P-450 monooxygenase system of hamster liver microsomes and its response to prior treatment with ethanol and other xenobiotics have been examined. Male Syrian golden hamsters were administered ethanol (ETOH), phenobarbital (PB), 5,6-benzoflavone (BF) or isoniazid (INH). Each treatment resulted in a moderate increase (20-60%) in the specific content of liver microsomal cytochrome P-450 along with a unique hemeprotein ferrous carbonyl Soret maximum. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of liver microsomes revealed distinctive changes in protein banding patterns in the cytochrome P-450 (45-60 kDa) region with each treatment. NADPH: cytochrome c reductase activity was increased by both PB and INH, whereas cytochrome b5 content was increased by INH only. Microsomal oxidation of ETOH and aniline p-hydroxylation (expressed per nmol cytochrome P-450) were enhanced dramatically by ETOH and INH, whereas PB and BF had no effect on these enzymatic activities. Both ETOH and INH also increased zoxazolamine 6-hydroxylation but, in contrast to other rodent species, this drug-metabolizing activity was decreased in hamster liver microsomes after treatment with either PB or BF. Microsomal benzphetamine N-demethylation was decreased by ETOH, INH and BF administration and was only modestly enhanced after treatment with PB. ETOH and INH had no effect on the O-deethylation of 7-ethoxycoumarin, and enzymatic activity increased by BF but decreased by PB. These results demonstrate that the cytochrome P-450-dependent monooxygenase system of hamster liver microsomes responds to treatment with ETOH and other xenobiotics in a manner that is quantitatively and, in certain respects, qualitatively different from that reported for the rat, rabbit, and mouse.

Animals↗
PubMed1987

Oxidative metabolism of carbon disulfide by isolated rat hepatocytes and microsomes.

The oxidative metabolism of carbon disulfide (CS2) was investigated in isolated rat hepatocytes and liver microsomes. In microsomes, CS2 metabolism was increased by phenobarbital pretreatment of the rats and decreased with pretreatment of the rats with cobaltous chloride. In both microsomes and hepatocytes, CS2 metabolism was inhibited by SKF-525A. Carbon dioxide (CO2) was the major volatile metabolite of CS2 in hepatocytes, and carbonyl sulfide (COS) was the major volatile metabolite in microsomal incubations. Addition of cytosol to microsomal incubations shifted the predominant volatile metabolite from COS to CO2 but did not change total volatile metabolite formation. Acetazolamide, a carbonic anhydrase inhibitor, significantly decreased COS metabolism but not CS2 metabolism in isolated hepatocytes or microsomes fortified with dialyzed cytosol. When [18O]H2O was included in incubations of microsomes and CS2, a substantial portion of the resulting COS was [18O] enriched, indicating that the oxygen atom was derived from water. These data are consistent with the hypothesis that CS2 is oxidized predominantly by the cytochrome P-450 containing monooxygenase system, and the product of this reaction is an unstable intermediate which reacts with water to form monothiocarbonate and reactive sulfur species. Monothiocarbonate is the hydrated form of COS. In intact hepatocytes, it is metabolized predominantly to CO2 and hydrogen sulfide. Unmetabolized monothiocarbonate can be dehydrated to COS. The majority of the reactive sulfur species and hydrogen sulfide are oxidized to nonvolatile sulfur compounds, including sulfate, but by different mechanisms.

Acetazolamide↗