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PubMed1975

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.

Animals↗

PubMed1976

Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody.

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

Animals↗

PubMed1986

Non-enzymic lipid peroxidation in microsomes and microsomal phospholipids induced by anthracyclines.

The stimulation of non-enzymic lipid peroxidation by doxorubicin, daunorubicin and 7 derivatives was investigated in extracted microsomal phospholipids and in intact microsomes. Evidence was obtained for the necessity of a free amino-sugar moiety for a stimulative effect on lipid peroxidation. Binding of anthracyclines to RNA (which is present in microsomes) was inhibitory towards stimulation. Drugs that stimulated lipid peroxidation in a non-enzymic system with extracted phospholipids also were stimulative in an enzymic, NADPH-dependent, microsomal system. They were not always effective in intact microsomes without the enzymic system. The role of the enzymic system in the stimulation of anthracycline induced lipid peroxidation is thought to be the reduction of iron ions rather than the stimulation of oxygen radical production via the anthracyclines.

Animals↗

PubMed1999

Suppression of cytosolic triacylglycerol recruitment for very low density lipoprotein assembly by inactivation of microsomal triglyceride transfer protein results in a delayed removal of apoB-48 and apoB-100 from microsomal and Golgi membranes of primary rat hepatocytes.

Cellular apoB in primary rat hepatocyte cultures was pulse-labeled with [(35)S]methionine for 1 h. Cells were then chased with excess unlabeled methionine for periods of up to 16 h in the presence or absence of BMS-200150, an inhibitor of microsomal triglyceride transfer protein (MTP). The secretion of apoB-48-VLDL was more sensitive to MTP inhibition than was apoB-100-VLDL. Inhibition of MTP had no inhibitory effect on the secretion of denser particles (apoB-48 HDL and apoB-100 HDL). BMS-200150 delayed the net removal of newly synthesized apoB-48 and apoB-100 from the microsomal and Golgi membranes, but not from the corresponding lumenal compartments. Only minor proportions of the microsomal lumen apoB-48 and apoB-100 (12-16% and 17-19%, respectively) were present as VLDL irrespective of whether MTP was inactivated or not. The HDL fraction contained most of the lumenal apoB-48 (67-73%) and a somewhat smaller proportion of apoB-100 (44-47%). The remainder of the lumenal apoB was associated with the IDL/LDL fraction. These proportions were unaffected by MTP inactivation. Excess labeled apoB which accumulated in the membranes in the presence of BMS-200150 was degraded. Inhibition of MTP prevented the removal of pre-synthesized triacylglycerol (TAG) from the hepatocytes as apoB-VLDL. Under these conditions intracellular TAG accumulated mainly in the cell cytosol, but also, to a lesser extent, in the microsomal membranes. The results suggest that inactivation of MTP inhibits a pathway of VLDL assembly which does not involve the bulk lumenal compartments of the microsomes. Suppression of this pathway ultimately prevents the net transfer of cytosolic TAG into mature apoB-VLDL.

Animals↗

PubMed1991

Neutral and acid retinyl ester hydrolases associated with rat liver microsomes: relationships to microsomal cholesteryl ester hydrolases.

We recently reported the presence of a neutral, bile salt-independent retinyl ester hydrolase (REH) activity in rat liver microsomes and showed that it was distinct from the previously studied bile salt-dependent REH and from nonspecific carboxylesterases (Harrison, E. H., and M. Z. Gad. 1989. J. Biol. Chem. 264: 17142-17147). We have now further characterized the hydrolysis of retinyl esters by liver microsomes and have compared the observed activities with those catalyzing the hydrolysis of cholesteryl esters. Microsomes and microsomal subfractions enriched in plasma membranes and endosomes catalyze the hydrolysis of retinyl esters at both neutral and acid pH. The acid and neutral REH enzyme activities can be distinguished from one another on the basis of selective inhibition by metal ions and by irreversible, active site-directed serine esterase inhibitors. The same preparations also catalyze the hydrolysis of cholesteryl esters at both acid and neutral pH. However, the enzyme(s) responsible for the neutral REH activity can be clearly responsible for the neutral REH activity can be clearly differentiated from the neutral cholesteryl ester hydrolase(s) on the basis of differential stability, sensitivity to proteolysis, and sensitivity to active site-directed reagents. These results suggest that the neutral, bile salt-independent REH is relatively specific for the hydrolysis of retinyl esters and thus may play an important physiological role in hepatic vitamin A metabolism. In contrast to the neutral hydrolases, the activities responsible for hydrolysis of retinyl esters and cholesterol esters at acid pH are similar in their responses to the treatments mentioned above. Thus, a single microsomal acid hydrolase may catalyze the hydrolysis of both types of ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1980

[Role of microviscosity of the lipid phase of microsomal membranes in regulation of enzymatic activity of glucose-6-phosphatase under microsomal modifications in vivo and in vitro].

The changes in microviscosity of the lipid phase of microsomal membranes under microsomal modification in vivo and in vitro were studied. It was shown that in intact microsome lipids there occur five thermo-induced structural transitions within the temperature range of 5--50 degrees. Delipidation of microsomes results in a shift in structural transitions temperature. Based on the literary and own data it was assumed that the breaks on the Arrhenius plots for glucose-6-phosphatase (EC 3.1.3.9) activity are due to phase-structural changes of microsomal lipids.

Animals↗

PubMed1984

[Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes].

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.

Animals↗

PubMed1997

Membranes of retinal microsomes: a new protein of the microsomal monooxigenase system.

A new component, which substitutes cytochrome P-450 as an acceptor of reducing equivalents from NADPH-cytochrome P-450 reductase, was identified in the bovine retina microsomal monooxigenase system, which does not contain cytochrome P-450. This component is a non-heme iron-containing protein with molecular mass of 66 kDa. The properties of the protein from the bovine retina are similar to those of MIP, a non-heme iron-containing protein from the heart microsomal monooxigenase system, in which cytochrome P-450 was not identified, either. Activation of the microsomal monooxigenase system (an increase in the NADPH-cytochrome P-450 reductase activity, an increased rate of microsomal NADPH oxidation) was shown in the retina upon long-term intensive illumination. It was shown also that the development of hereditary degeneration of the retina in rats was accompanied by activation of the specific microsomal monooxigenase system in the target tissues (retina, brain cortex) irrespective of its composition (cytochrome P-450 or non-heme iron-containing protein).

Animals↗

PubMed1982

Regulation by Lipids of Plant Microsomal Enzymes: III. Phospholipid Dependence of the Cytidine-Diphospho-Choline Phosphotransferase of Potato Microsomes.

Cytidine-diphospho-choline diacyl-glycerol phosphorylcholine phosphotransferase activity was demonstrated in potato (Solanum tuberosum L.) microsomes and the incorporation of cytidine-diphospho[(14)C]choline into phosphatidylcholine was characterized by the time course of (14)C incorporation and the effect of microsomal protein concentration on choline incorporation.Potato microsomes were progressively delipidated by treatments (2 min at 0 degrees C) with increasing amounts of phospholipase C from Bacillus cereus. A decrease in choline phosphotransferase activity was observed in parallel with the progressive hydrolysis of membrane phospholipids. A 70% (or more) phospholipid hydrolysis provoked the total inactivation of the enzyme.Adding back exogenous phospholipids (in the form of liposomes) to phospholipase C-treated membranes restored the enzymic activity. Restoration could be obtained with egg yolk phospholipids as well as with potato phospholipids. Restoration was time dependent and completed after 10 minutes; restoration was also dependent on the quantity of liposomes added to lipid-depleted membranes: the best restorations were obtained with 1 to 2.5 milligrams of phospholipid per mg of microsomal protein; higher phospholipid to protein ratios were less efficient or inhibitory.These results clearly demonstrate the phospholipid dependence of the cytidine-diphospho-choline phosphotransferase from potato microsomes.

Journal Article↗

PubMed1993

Tamoxifen inhibits lipid peroxidation in cardiac microsomes. Comparison with liver microsomes and potential relevance to the cardiovascular benefits associated with cancer prevention and treatment by tamoxifen.

Tamoxifen and 4-hydroxytamoxifen were both good inhibitors of iron-dependent lipid peroxidation in rat cardiac microsomes. Tamoxifen was also a good inhibitor of lipid peroxidation in liposomes prepared from the phospholipid obtained from rat liver microsomes. In a modified rat liver microsomal system containing a sufficiently low amount of peroxidizable phospholipid to make it comparable with the rat cardiac microsomal system, tamoxifen and 4-hydroxytamoxifen were of similar effectiveness as in the cardiac system. Tamoxifen is known to lower serum cholesterol levels, and the findings reported here indicate that the drug might also protect heart cell membranes against peroxidative damage. Potential cardioprotective and antiatherosclerotic benefits of tamoxifen are discussed in relation to the drug's use in cancer prevention and treatment.

Animals↗

PubMed2005

In vitro studies in microsomes from rat and human liver, kidney, and intestine suggest that perfluorooctanoic acid is not a substrate for microsomal UDP-glucuronosyltransferases.

Perfluorooctanoic acid (PFOA) is a fluorinated fatty acid analogue used as a surfactant in the manufacture of fluoropolymers. Previous studies have indicated that PFOA was metabolically inert in mammals, but recent metabolism studies with related fluorochemicals suggested that PFOA might form a glucuronide conjugate. [(14)C(1)]-PFOA was incubated with male and female human and rat liver, kidney, and small intestine microsomes. Incubations were carried out in the presence of alamethicin and beta-saccharolactone to increase access of PFOA to the enzyme active site and to inhibit potential hydrolysis of PFOA-glucuronide by microsomal beta-glucuronidase, respectively. Although positive control experiments using p-nitrophenol demonstrated significant UDP-glucuronosyltransferase (UDPGT) activity in all of the tested microsomal preparations, no evidence for formation of a PFOA-glucuronide was obtained, either by high-sensitivity radiochromatography or by LC/MS. These data suggest that PFOA is not a substrate for human or rodent microsomal UDPGTs.

Animals↗

PubMed1996

Detection on immunoblot of new proteins from the microsomal fraction recognized by anti-liver-kidney microsome antibodies type 1.

Previous studies have demonstrated that sera from patients with autoimmune hepatitis type 1 contain antibodies which react with proteins other than the endoplasmic reticulum integral membrane protein of apparent Mr 50,000, now known to be a cytochrome P450 of the IID subfamily. Sera from 141 patients found by immunofluorescence to be positive for anti-liver-kidney microsome antibodies type 1, and sera from 50 blood donors used as controls, were analyzed by immunoblotting experiments on rat liver microsomes, microsomal subfractions, and also microsomes subjected to various treatments, as described in the text. These fractions were characterized morphologically by electronic microscopy and biochemically by different enzymatic activities. Five bands were found to be stained more often by the patients' sera than by the controls' and with a statistically significant difference in frequency. These antigenic proteins were located at apparent Mr 62,000, 58,000, 50,000, 40,000, and 35,000. The 50,000 protein was of course more often stained than the others. Antibodies against these antigens belonged essentially to the IgG1 subclass. For some of them, subcellular localization and membrane topography are discussed. Interestingly, the 58,000 protein is not an integral membrane protein.

Adult↗

PubMed1984

Mitochondrial-microsomal interactions in vitro: use of microsomal 7-ethoxycoumarin O-deethylase to study succinate-stimulated malate efflux from mitochondria.

An assay system was developed utilizing microsomal 7-ethoxycoumarin O-deethylase to monitor the formation and efflux of malate from mitochondria in vitro. Malate was coupled by means of malic enzyme, NADP+, and the microsomal mixed-function oxidase system to the production of highly fluorescent 7-hydroxycoumarin. Under conditions of the assay, steady-state concentrations of NADPH could be maintained, and half-maximal rates of microsomal 7-ethoxycoumarin O-deethylase were observed with about 0.2 microM NADPH and 250 microM malate. Addition of succinate to mixtures of intact mitochondria and microsomes enhanced rates of 7-hydroxycoumarin formation by facilitating the generation and transport of malate from mitochondria (half-maximal stimulation was observed with 0.4 mM succinate). Stimulation of 7-hydroxycoumarin formation by succinate, under conditions where concentrations of cofactors and substrate were adjusted to resemble those in the intact cell, provides further evidence that reducing equivalents for mixed-function oxidation may arise via malate efflux from mitochondria.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1984

Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes.

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.

Animals↗

PubMed1984

Isopycnic centrifugation of rat-liver microsomes in isoosmotic gradients of Percoll and release of microsomal material by low concentrations of sodium deoxycholate.

Rat-liver microsomes were subjected to isopycnic centrifugation under isoosmotic conditions in gradients of Percoll containing 0%, 0.05%, 0.1% and 0.2% sodium deoxycholate, respectively. The buoyant density of the microsomes was in the range 1.043 to 1.046 g/ml, independent of the detergent concentration. Absorbance measurements and polyacrylamide gel electrophoresis revealed that the detergent causes an increase in the release of material from the microsomes. Electron microscopy studies showed that membrane disassembly was avoided if the microsomes were isolated at concentrations below 0.1% sodium deoxycholate.

Animals↗

PubMed1975

Regulation of microsomal enzymes by phospholipids. IX. Production of uniquely modified forms of microsomal UDP-glucuronyltransferase by treatment with phospholipase A and detergents.

The kinetic parameters of microsomal UDPglucuronyltransferase (EC 2.4.1.17) were compared after treatment of microsomes with phospholipase A and Triton X-100. Treatments with phospholipase A and Triton had differential effects on activities at V (assayed in both forward and reverse directions), the affinity of the enzyme for UDPglucuronic acid and UDP, the specificity of binding of UDPsugars, and the sensitivity of the enzyme to treatment with UDP-N-acetylgulcosamine. Comparisons of lysophosphatide, Triton, and cholate-activated forms indicated that different types of detergents activate by separate mechanisms. These data are taken as evidence that selective types of changes of the composition and structure of microsomal lipids lead to selective types of changes of the properties of UDPglucuronyltransferase. Activation of UDPglucuronyltransferase in response to different types of perturbations of the membrane lipids is thus a specific process. It was found that constraint on the maximal potential activity of the reverse reaction catalyzed by UDPglucuronyltransferase is more extensive in untreated microsomes than constraint on the forward reaction. Also, the "activated forms" of UDPglucuronyltransferase have less activity than the untreated enzyme if assays are carried out under conditions presumed to exist in vivo. These results indicate the complexity of lipid-protein interactions as regulators of membrane-bound enzymes. They also indicate the functional significance of this type of regulation for the activity of UDPglucuronyltransferase.

Animals↗

PubMed1986

The activity of 3-hydroxy-3-methylglutaryl-CoA reductase and acyl-CoA: cholesterol acyltransferase in hepatic microsomes from male, female and pregnant rats. The effect of cholestyramine treatment and the relationship of enzyme activity to microsomal lipid composition.

The relationship of microsomal cholesterol and phospholipid fatty acid composition to the activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA: cholesterol acyltransferase was investigated in male, female virgin and pregnant rats when hepatic cholesterogenesis was stimulated by cholestyramine. Cholestyramine increased HMG-CoA reductase activity in both sexes but had no effect on microsomal free cholesterol level or acyl-CoA: cholesterol acyltransferase activity. The data suggest that during cholestyramine treatment high rates of bile acid synthesis are supported by preferential channelling of cholesterol into this pathway, whilst the substrate pool and activity of acyl-CoA:cholesterol acyltransferase are maintained unaltered. The lack of a consistent relationship among enzyme activities and microsomal lipid composition infers that HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase are regulated in vivo by independent mechanisms which are unlikely to involve modulation by the physical properties of the microsomal lipid.

Animals↗

PubMed1992

Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of delta 9-tetrahydrocannabinol by guinea pig liver microsomes.

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.

Amino Acid Sequence↗