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PubMed1986

Enzymes of the purine metabolism in rat brain microsomes.

Rat brain microsomes, when they are suspended in moderate ionic strength medium, released enzyme activities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), adenosine deaminase (ADA, E.C.3.5.4.4), guanine deaminase (GAH, E.C.3.5.4.3), and purine nucleoside phosphorylase (PNP, E.C.2.1.2.4). The activities released decreased when the saline concentration of the medium was increased and the opposite occurred when 50 mM, pH 7.4 sodium phosphate medium was used. Rat brain microsomes that had been extracted previously by moderate ionic strength solutions still had activities of all the enzymes tested, and released these activities upon sonication or deoxycholate (DOC) treatment. The proportion of the activity released was similar for all the enzymes. DOC treatment released higher enzymic activities and a smaller amount of protein than sonication did. The proportion of activities released was similar to that found in the 105,000 g supernatant. The suspension of microsomes still retained activities of the above-mentioned enzymes after consecutive extractions with increasing concentrations of detergent solutions (DOC and Triton X-100). The amount of enzymic activities released from the microsomes by sonication or DOC treatment did not depend on the protein composition of the homogenization medium. Thus, on increasing the enzyme concentration in the homogenization medium, the activities released did not increase in parallel. The set of results obtained showed that the microsomal fraction is as useful as the cytosolic one for studying purine catabolism in rat brain. Furthermore, the conditions in which purine enzymes are attached to the microsomal fraction are probably closer to "in vivo" conditions than those in which these enzymes are found in the soluble fraction.

Animals↗

PubMed1991

The possible role of phospholipase A2 in hepatic microsomal lipid peroxidation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats.

The induction of lipid peroxidation in hepatic microsomes of rodents treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is well documented. The potential mechanisms involved in TCDD-induced microsomal lipid peroxidation were investigated, using selected inhibitors and free radical scavengers in vitro. Rats were treated with 40 micrograms TCDD/kg orally as a single dose. Inhibitors of phospholipase A2, including a variety of phenothiazines, dibucaine, imipramine, and verapamil, inhibited in vitro microsomal lipid peroxidation in response to TCDD administration. In addition, the lipoxygenase inhibitor quercetin, and the hydrogen peroxide scavenger aminopyrine inhibited lipid peroxidation with microsomes from TCDD-treated rats. The singlet oxygen scavenger beta-carotene, the cytochrome P-450 substrate benzphetamine, and the cyclooxygenase inhibitor indomethacin produced moderate enhancement of hepatic microsomal lipid peroxidation. The results suggest that activation of phospholipase A2 may play a critical role in the metabolic events associated with hepatotoxicity and ultimate cell death produced by TCDD. The results also support the involvement of hydrogen peroxide in TCDD-induced microsomal lipid peroxidation.

Aminopyrine↗

PubMed1994

Lipid peroxidation in microsomes of murine bone marrow after low-dose gamma-irradiation.

The principal aim of the study was to investigate the effect of low-dose gamma-irradiation on lipid peroxidation (LPO) in murine bone marrow. To this end, the degree of LPO in suspensions of microsomes of murine bone marrow cells (BMC) was determined in terms of malondialdehyde (MDA) formation after whole-body or in vitro exposure to various doses of gamma-radiation. These effects were compared to some extent with similar effects in liver and spleen preparations. As to the effect of gamma-irradiation on LPO in BMC, the response depends on the dose level and on whether whole-body or in vitro exposures are involved. Whole-body irradiation did not result in an increase in LPO in BMC microsomes, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of BMC microsomes with 0.1, 10 and 50 Gy brought about an increase in LPO. This increase was already significant (P < 0.05) at 0.1 Gy following a post-irradiation incubation and substantial at 50 Gy, even without subsequent incubation. The results show that low doses of gamma-irradiation are able to induce an elevation of LPO in murine BMC microsomes, but only after in vitro irradiation. In the case of whole-body irradiation cellular radical scavengers and other metabolic reactions may prevent a measurable increase in LPO. This is partly illustrated by the case of vitamin-E deficiency, where a substantial increase in LPO in BMC microsomes is observed even without gamma-irradiation in comparison with euvitaminotic mice because normally occurring radicals are not scavenged sufficiently.

Animals↗

PubMed1983

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes.

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.

Adenosine Triphosphate↗

PubMed1991

Kinetic studies on toluene metabolism in ethanol- and phenobarbital-induced rat liver microsomes in vitro.

In vitro metabolism of toluene was investigated at substrate concentrations of 0.03-6.25 mM in liver microsomes from control and ethanol- and phenobarbital (PB)-treated rats. Three metabolites, benzylalcohol (BA), o- and p-cresol, were measured by high-performance liquid chromatograph. BA was the main metabolite of toluene, whereas o- and p-cresol contributed only 1.1-1.5% and 1.7-2.8% of total metabolites, respectively, in microsomes from control rats. Ethanol treatment showed little effect on the percentages of three metabolites, but PB increased the percentages of o- and p-cresol to as high as 5.5% and 8.0%, respectively, following the increase in toluene concentration. There were two different isozymes with different Km involved in the side-chain hydroxylation of toluene in microsomes from control and ethanol-treated rats. One had a low Km value (0.13-0.17 mM) and could be greatly induced with ethanol treatment. The other was a high Km isozyme (0.60-0.87 mM). PB-induced isozyme showed a similar Km value to that of the high Km isozyme existing in microsomes from control and ethanol-treated rats. Two isozymes were involved in the formation of p-cresol in microsomes of control rats: the low-Km type had a similar value (0.15 mM) to the low isozyme of BA formation, but the high Km isozyme had a larger value (2.04 mM) than the high isozyme of BA. Only one enzyme responsible for o-cresol formation was detected in microsomes of control rats, and had a similar Km (2.11 mM) to that of the high Km isozyme of p-cresol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1975

Dietary and hormonal effects upon activity of "soluble" protein and particulate fraction of fatty acid desaturation system of rat liver microsomes.

Rat liver microsomes were extracted with a buffered 0.15 M KCl and 0.25 M sucrose solution and fractionated by centrifugation into a particulate component and a supernatant containing a protein factor necessary for fatty acid desaturation. The delta 6 fatty acid desaturation activity of the extracted microsomes was reduced significantly, and the readdition of the supernatant restored the enzymatic activity to the original value of the whole microsomes. A protein diet or a fat-free diet increased the delta 6 desaturation activity of the whole microsomes. The activating effect was evoked upon the particulate components of the enzymatic desaturation system and not upon the protein factor present in the supernatant. Fasting, refeeding, and refeeding plus glucagon and theophylline treatments of rats also modified the delta 6 desaturation activity of whole liver microsomes. The effect also was evoked on the delta 6 desaturation system tightly bound to the microsomal membrane but not on the protein factor of the supernatant. Accordingly, the protein factor of the supernatant is considered to be different from the cyanide sensitive factor and the desaturase.

Animals↗

PubMed1976

Effect of bovine serum albumin on monoacyl- and diacylglycerol 3-phosphate formation in mitochondrial and microsomal fractions of rabbit hearts.

The formation of monoacyl- and diacylglycerol 3-phosphate (P) by rabbit heart mitochondrial and microsomal fractions was studied by varying the concentration of acyl-CoA and that of bovine serum albumin in the assay system. The two subcellular fractions were prepared by the conventional differential centrifugation technique. The optimal concentration of acyl-CoA for both mitochondrial and microsomal acylation of glycerol 3-P was shifted to a higher range of acyl-CoA concentrations by greater amounts of albumin. A similar shift in the acyl-CoA concentration-enzyme activity relationship was observed in the acylation reaction of 1-palmitoylglycerol 3-P by the heart microsomes. The addition of albumin increased slightly the rate of diacylglycerol 3-P accumulation but increased greatly the rate of monoacylglycerol 3-P accumulation at any concentration of acyl-CoA; the effect was observed with mitochondrial or microsomal fraction as the crude enzyme source. Moreover, palmitoyl-CoA and linoleoyl-CoA served equally well as the acyl donor for the acylation reaction. However, relatively more monoacyl- than diacylglycerol 3-P was accumulated in the assays with rabbit heart mitochondrial fraction in the presence of albumin, whereas more diacyl- than monoacylglycerol 3-P was formed by the microsomal fraction. As a result, the microsomal diacyl:monoacyl-glycerol 3-P ratio was invariably greater than the mitochondrial ratio at a given concentration of acyl-CoA and albumin.

Animals↗

PubMed1982

The lipid composition of human liver microsomes.

The lipid composition of human liver microsomes isolated from liver biopsy samples obtained at abdominal surgery has been determined. Human liver microsomal phospholipid is composed of 49% phosphatidylcholine, 31% phosphatidylethanolamine, 14% phosphatidylserine + phosphatidylinositol and 6% sphingomyelin, very similar to the phospholipid composition of rat liver microsomes. The fatty acid composition of human liver microsomes is remarkable only for its content of polyunsaturated fatty acids, with 20% of the fatty acids consisting of arachidonic, docosatetraenoic, docosapentaenoic and docosahexaenoic acids. This value contrasts with 33% in rats and 9% in rabbits. The molar cholesterol/phospholipid ratio in human liver microsomes is 0.069, similar to the ratio in rat and rabbit microsomes.

Aged↗

PubMed1991

Effect of dietary fat on diabetes-induced changes in liver microsomal fatty acid composition and glucose-6-phosphatase activity in rats.

Experimental diabetes may manifest itself in a defect in liver microsomal fatty acid desaturation and increased activity of glucose-6-phosphatase (G-6-Pase). The present study was designed to determine whether these changes could be normalized by a change in the dietary fat consumed. Control and streptozotocin-induced diabetic rats were fed nutritionally adequate diets which varied in fatty acid composition. Fatty acid analysis of liver microsomal phospholipids revealed that non-diabetic control animals fed saturated fat (beef tallow) or a diet high in omega 3 fatty acids (fish oil) exhibited a significantly higher level of 18:2 omega 6 and a lower level of 20:4 omega 6 in the phosphatidylcholine and phosphatidylethanolamine fractions compared with diabetic animals. Control and diabetic animals fed the high linoleic acid diet had similar levels of 18:2 omega 6 in the microsomal phosphatidylcholine and phosphatidylserine fractions. Microsomal G-6-Pase activity was higher in diabetic than in control animals. Activity of G-6-Pase was lower in microsomes of control animals fed the soybean oil or the fish oil diet, but was not significantly reduced in diabetic animals fed high polyunsaturated fats. Blood glucose levels were similar in control groups fed the different diets, but the plasma hemoglobin Alc level was lower in diabetic animals fed the soybean oil diet. Cholesterol and triglyceride levels were lower in diabetic animals fed the fish oil-based diet. The results suggest that dietary fat manipulation has the potential to change at least some of the abnormalities in the microsomal membrane in experimental diabetes.

Animals↗

PubMed1988

Effects of aurothioglucose on iron-induced rat liver microsomal lipid peroxidation.

Aurothioglucose (ATG), an inhibitor of selenium-dependent glutathione peroxidase activity, at a concentration of 100 microM, strongly increases lipid peroxidation of rat liver microsomes exposed to either ferrous ion (10 microM) or the combination of ferric ion (10 microM) and ascorbic acid (500 microM), in the presence of reduced glutathione (GSH, 800 microM). This effect was not achieved using heat-inactivated microsomes and was dependent on the presence of GSH. ATG did not affect the lag period associated with ascorbic acid/ferric ion-induced microsomal lipid peroxidation (previously attributed to an undefined GSH-dependent microsomal agent), but did increase the rate of peroxidation subsequent to the lag period. The potent GSH-dependent inhibition of microsomal lipid peroxidation by cytosol (10% of total volume) was completely reversed by ATG (100 microM). ATG similarly reversed an inhibition of phosphatidylcholine hydroperoxide-dependent liposomal peroxidation that has been attributed to phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme distinct from the classical glutathione that cannot utilize intact phospholipids. ATG inhibited, in addition to the classical selenium-dependent glutathione peroxidase, both cytosolic and microsomal (basal and N-ethyl maleimide-stimulated) glutathione S-transferase activities with greater than 80% inhibition achieved at 100 microM ATG. ATG, at concentrations up to 250 microM, did not inhibit PHGPX activity measured by the coupled-enzyme method in the presence of Triton X-100 (0.1%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Hydroxylation of fatty acids and alcohols by hepatic microsomal cytochrome P-450 system from the Mongolian gerbil.

The liver microsomes of the Mongolian gerbil Meriones unguiculatus catalyzed the hydroxylation of various saturated fatty acids (C8-c18), alcohols (C12 and C16) and hydrocarbon (C12) to the corresponding omega- and (omega-1)-hydroxy derivatives. Lauric acid was hydroxylated most effectively among saturated fatty acids and the order of activity as hydroxylation substrates was C12 greater than C14 greater than C13 greater than C16 greater than C10 greater than C18 greater than C8. The specific activity of laurate hydroxylation (5.99 nmol/mg microsomal protein/min) in gerbil liver microsomes was higher than that observed in other species. 1-Dodecanol was also hydroxylated very effectively (4.58 nmol/mg microsomal protein/min) by gerbil liver microsomes, but in general the hydroxylation rates for fatty alcohols were much lower than those for the corresponding acids. It was found from both inhibitor and cofactor studies that the enzyme catalyzing the hydroxylation of fatty acids and alcohols in the liver microsomes of the Mongolian gerbil was a typical cytochrome P-450-linked monooxygenase, and at least two different cytochrome P-450 species were involved in the hydroxylation.

Animals↗

PubMed1976

Biosynthesis of molecular species of CDP-diglyceride from endogenously-labeled phosphatidate in rat liver microsomes.

The biosynthesis of [14C] CDP-diglyceride was studied using rat liver microsomes which were endogenously labeled with [14C] phosphatidic acid by preincubation of unlabeled microsomes with sn-[14C] glycerol-3-phosphate and appropriate cofactors. The formation of CDP-diglyceride from radioactive phosphatidate showed an absolute requirement for CTP and MgCl2. The newly formed [14C] CDP-diglyceride was characterized by thin layer chromatography (TLC), isotopic labeling from radioactive CTP, and its ability to serve as substrate for the microsomal enzyme, CDP-diglyceride: inositol phosphatidyltransferase. The distributions of radioactive glycerol-3-phosphate among the various chemical classes of microsomal [14C] phosphatidate and [14C] CDP-diglyceride were determined following argentation TLC of their 1,2-diglyceride acetate derivatives. Most of the radioactivity among the phosphatidic acids was present in the monoenoic (36%) and dienoic (33%) molecular species, whereas 10, 8, 4, and 8% were associated with the saturates, trienes, tetraenes, and polyenes, respectively. Similar distributions of radioactivity were found among the corresponding classes of newly formed CDP-diglyceride. Only a slight enrichment of radioactivity in the tetraenoic CDP-diglyceride was found relative to the corresponding phosphatidates. Therefore, under the conditions of study, the microsomal CTP: phosphatidate cytidylyltransferase produces mainly monoenoic and dienoic species of CDP-diglyceride and shows little specificity towards different molecular species of phosphatidic acids. The present results suggest also that the arachidonoyl phosphatidate derived from the microsomal acylation of sn-glycerol-3-phosphate is not likely the major source of arachidonic acid in liver phosphatidylinositol.

Animals↗

PubMed1990

The effect of glutathione on the vitamin E requirement for inhibition of liver microsomal lipid peroxidation.

Vitamin E dependent inhibition of rat liver microsomal lipid peroxidation in an NADPH and ADP-Fe+3 containing system occurred at lower vitamin E concentrations in the presence of glutathione (GSH). Using microsomes from rats fed a vitamin E deficient diet, vitamin E was shown to be required for inhibition. Inhibition also required the presence of a storage labile microsomal component, since no inhibition was observed when using microsomes that had been stored for one month. This observation provides evidence that direct reduction of reversibly oxidized vitamin E by GSH does not appear to contribute significantly to inhibition of peroxidation. During GSH and vitamin E dependent inhibition of lipid peroxidation, vitamin E (reduced form) concentrations remained constant, indicating that GSH maintained vitamin E concentrations. Without GSH, vitamin E concentrations dropped rapidly. By adding vitamin E to microsomes, it was found that inhibition of lipid peroxidation in the presence of GSH occurred at about five-fold less vitamin E than in the absence of GSH. Inhibition at these lower levels of vitamin E was 85-90% complete. Results indicate that GSH can be used to maintain vitamin E (reduced form) concentrations, thereby lowering the concentration of vitamin E necessary to inhibit microsomal lipid peroxidation.

Adenosine Diphosphate↗

PubMed1992

A comparative study of the effects of cadmium on microsomal mixed-function oxidase activities between kidney cortex and liver.

The effects of cadmium and phenobarbital (PB) administration on the activities of microsomal mixed-function oxidase (MFO) in rat kidney cortex and liver were studied. Five indices, including cytochrome P-450 contents, aniline hydroxylase, NADPH-cytochrome c reductase, aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase activities, were assayed. By comparison, it was found that PB could induce MFO activities in kidney cortex microsomes and liver microsomes, both hepatic and renal microsomal MFO were decreased by the ip injection of CdCl2, and Cd2+ could markedly decrease the phenobarbital induction to hepatic and renal microsomal MFO activities with the exception of NADPH-cytochrome c reductase in kidney cortex microsomes.

Animals↗

PubMed1989

The effects of cadmium, mercury and lead in vitro on hepatic microsomal mixed function oxidase and lipid peroxidation.

In this study, the authors investigated the effect of cadmium, mercury and lead in vitro on hepatic microsomal mixed function oxidase and lipid peroxidation. The results showed that these metals could inhibit the activity of hepatic microsomal aniline hydroxylase, decrease the concentration of hepatic microsomal cytochrome P-450 and increase the concentration of the inactive form of hemoprotein, P-420. Besides, they could enhance hepatic microsomal lipid peroxidation. There were marked concentration-dependent responses in these microsomal reactions. Thus, it may be suggested that cadmium, mercury and lead are capable of impairing hepatic microsomal mixed function oxidase in vitro by stimulating membrane lipid peroxidation.

Aniline Hydroxylase↗

PubMed1980

Effect of metal salts on UDPglucuronosyltransferase activity of various aglycones in rat liver microsomes in vitro.

The effects of aluminium, manganese, ferrous and ferric iron, cobalt, nickel, copper, zinc, cadmium, barium, mercury and lead ions on the rat hepatic UDPglucuronosyltransferase activity with regard to 4-methylumbelliferone, 4-nitrophenol, bilirubin, and 2-aminophenol were investigated in vitro. Conjugation of bilirubin was found to be activated by manganese, cobalt, nickel, cadmium, mercury and lead ions in native microsomes, but not in digitonin-treated microsomes. Aluminum, ferrous iron, cadmium, barium and lead increased the conjugation of 4-nitrophenol in native microsomes and aluminium in digitonin-treated microsomes as well. Lead slightly activated 4-methylumbelliferone conjugation, whereas none of the metal salts studied were found to activate the conjugation of 2-aminophenol. Copper and zinc salts inhibited all conjugations. In digitonin-treated microsomes, the conjugation of 4-nitrophenol was inhibited by cadmium and mercury, that of 4-methylumbelliferone by nickel and lead as well. The sensitivity of 4-methylumbelliferone conjugation to inhibition by mercury was most pronounced in microsomes prepared from rats pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Aminophenols↗

PubMedDate not supplied

Metabolic assessment in liver microsomes by co-activating cytochrome P450s and UDP-glycosyltransferases.

A "dual-activity" microsomal system in which both CYPs and UGTs were active was evaluated for studies of metabolic stability and in-vitro metabolite profiling. In this "dual-activity" system, alamethicin, a pore-forming peptide, was used to activate UGTs in human liver microsomes without affecting CYP activity. Interference studies indicated that CYP cofactors had little effect on UGT surrogate activity as measured by glucuronidation of acetaminophen and trifluoperazine. Further, UGT cofactor, UDPGA (< 2 mM), did not inhibit the marker activity of five major CYPs including 1A2, 2C9, 2C19, 2D6 and 3A4, suggesting that both oxidation and glucuronidation can be co-activated in microsomes. In a comparison study, compounds with significant glucuronidation showed distinct stability profiles in the "dual-activity" system, compared to the conventional microsomal incubation in which only CYPs were active. For compounds with minor or no glucuronidation, the metabolic stability remained similar between the "dual-activity" system and the conventional microsomal incubation. The feasibility of this "dual-activity" system utilized for metabolite profiling was also investigated using tramadol as a model drug. It was found that oxidative metabolites of tramadol generated in the "dual-activity" system matched those detected in the conventional microsomal incubation. However, tramadol glucuronide was observed in the "dual-activity" system but not in the conventional micromosal incubation. Results clearly suggest that the "dual-activity" system is a valuable in vitro model for metabolism studies in drug discovery.

Alamethicin↗

PubMed2003

Paclitaxel metabolism in rat and human liver microsomes is inhibited by phenolic antioxidants.

Paclitaxel is an important, recently introduced anti-neoplastic drug. Paclitaxel metabolites are virtually inactive in comparison with the parent drug. The study investigated whether phenolic antioxidants could inhibit metabolic inactivation sufficiently to increase paclitaxel effects. Cytochrome p450 (CYP)-catalysed metabolism of paclitaxel was investigated in rat and human liver microsomes. In rat microsomes, paclitaxel was metabolised mainly to C3'-hydroxypaclitaxel (C3'-OHP), less to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP) and another monohydroxylated paclitaxel. In human liver microsomes, 6alpha-hydroxypaclitaxel (6alpha-OHP), formed by CYP2C8, was the main metabolite, while C3'-OHP, C2-OHP and another product different from di-OHP were minor metabolites, formed by CYP3A4. In individual human livers 6alpha-OHP was formed at 1.8-fold to 13-fold higher rates than C3'-OHP. Kinetic parameters (K(m) and V(max)) of production of various metabolites in rat and human liver microsomes revealed differences between species as well as human individual differences. Nine phenolic antioxidants ((+)-catechin, (-)-epicatechin, fisetin, gallic acid, morin, myricetin, naringenin, quercetin and resveratrol) were tested for inhibition of paclitaxel metabolism. In rat microsomes, resveratrol was more inhibitory than fisetin; the other phenolic antioxidants were without effect. In human microsomes, the inhibiting potency decreased in the order fisetin >quercetin >morin >resveratrol, while the other phenolic antioxidants were not inhibitory; the formation of 6alpha-OHP (CYP2C8) was generally more inhibited than that of C3'-OHP. The inhibition was mostly mixed-type. The results suggest that oral administration of some phenolic substances might increase paclitaxel blood concentrations during chemotherapy.

Adolescent↗