Rat Liver Microsomal Phospholipid Research Articles

Microsomal glutathione transferase: Lipid-derived substrates and lipid dependence

Rat liver microsomal glutathione transferase was found to display glutathione peroxidase activity toward a variety of oxidized lipids. 1-Linoleoyl-2-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylethanolamine hydroperoxide, and cholesteryl linoleate hydroperoxide all served as substrates (0.02, 0.04, 0.02, and 0.02 μmol/min mg, respectively). The phospholipid hydroperoxide glutathione peroxidase activity of the enzyme was found not require detergent and increased when liposomes containing peroxidized phospholipid were fused with liposomes containing microsomal glutathione transferase. Methyl linoleate ozonide serves as a very efficient substrate for the microsomal glutathione transferase. The unactivated and N-ethylmaleimide-activated enzyme displayed specific activities of 0.74 and 5.9 μmol/min mg, respectively. Upon examination of a series of 4-hydroxyalk-2-enals it was found that the catalytic efficiency of the enzyme increases from the 4-hydroxyhept-2-enal up to the 4-hydroxytetradec-2-enal. The specific activities with the various 4-hydroxyalk-2-enals tested varied between 0.28 and 0.95 μmol/min mg. The phospholipid dependence of the microsomal glutathione transferase was examined in proteoliposomes formed by cholate dialysis. Phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, and rat liver microsomal phospholipids could all be used successfully to reconstitute the enzyme. In conclusion, microsomal glutathione transferase can detoxify a number of lipid peroxidation products as well as fatty acid ozonide. The results imply a protective role for the enzyme under conditions of oxidative stress.

Effects of Oral Administration of Acitretin on Rat Liver Microsomal Phospholipids, P-450 Content and Monooxygenase Activities

Doses of 3 and 10 mg/kg/day acitretin were orally administered to female Wistar rats over a period of 6 weeks. Phospholipid classes, P-450 content, aminopyrine-N-demethylase (ADM) and 7-ethoxyresorufin-O-deethylase (7-ERO-D) activities were determined in the liver microsomes of the treated animals. Both dosages caused statistically significant alterations in rat liver microsomal phospholipid composition which may be associated with changes in the metabolic activity, ionic transport, cell-cell interaction and other processes of the hepatic cellular components. Furthermore, statistically significant alterations of P-450 isozyme activities were induced by both dosages. Our data suggest a possible interaction of acitretin with other drugs and endogenous substances metabolized by these enzyme systems.

The purification and molecular weight determination of rat liver microsomal phospholipid N-methyltransferase

Conference Article| November 01 1993 The purification and molecular weight determination of rat liver microsomal phospholipid N-methyltransferase William A. Gibbons; William A. Gibbons 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Luma Auchi; Luma Auchi 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Kevin McBride; Kevin McBride 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Alfred N. Fonteh; Alfred N. Fonteh 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Banafshe Larijani; Banafshe Larijani 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Vadim Tsvetnitsky; Vadim Tsvetnitsky 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Margaret M. Anderson; Margaret M. Anderson 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Francis A. Yeboah Francis A. Yeboah 1University-Industry Centre for Pharmaceutical Research, School of Pharmacy, University of London, 29–39 Brunswick Square, London WC1N 1AX Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1993) 21 (4): 449S. https://doi.org/10.1042/bst021449s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation William A. Gibbons, Luma Auchi, Kevin McBride, Alfred N. Fonteh, Banafshe Larijani, Vadim Tsvetnitsky, Margaret M. Anderson, Francis A. Yeboah; The purification and molecular weight determination of rat liver microsomal phospholipid N-methyltransferase. Biochem Soc Trans 1 November 1993; 21 (4): 449S. doi: https://doi.org/10.1042/bst021449s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1993 Biochemical Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.

Lipid Peroxidation of Microsomal Membranes as the Mechanism of 4-Aminopyrazolopyrimidine-Induced Cell Injury in the Rat Liver.

The effect of 4-aminopyrazolo [3, 4-d]pyrimidine (4-APP) on lipid peroxidation in rat liver was characterized. In 4-APP (50mg/kg)-treated rats, the amount of lipid peroxides increased from 150 to 380nmol/g of liver. The action of 4-APP on lipid peroxidation was investigated in an in vitro system containing rat liver microsomal phospholipid liposomes, NADPH, purified microsomal NADPH cytochrome c reductase, and Fe3+-ADP complexes. The addition of 4-APP resulted in the enhancement of lipid peroxidation monitored by O2 consumption and the peroxidative cleavage of unsaturated phospholipids. Superoxide dismutase, the iron chelator desferoxaminemethanesulfonate, and ceruloplasmin inhibited the 4-APP-induced lipid peroxidation. In contrast, catalase and sodium benzoate, which are known to scavenge hydrogen peroxide and hydroxyl radicals, failed to prevent phospholipid peroxidation. These findings strongly suggest that the enzymatic reduction of Fe3+-ADP-4-APP complexes by NADPH cytochrome c reductase significantly promoted lipid peroxidation in microsomal phospholipid membranes, and was probably mediated by an active iron-oxygen complex.

Deacylation of endogenously deuterated rat liver microsomal phospholipids by endogenous phospholipases

The relative deacylation of microsomal phospholipid molecular species was reexamined. Microsomal membranes were prepared from the livers of rats injected, over a period of 20 h, with perdeuterated ethanol. The phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography of the total lipid extracts and the distribution of deuterium among the molecular species of the diacylglycerol moieties was determined by reversed-phase high pressure liquid chromatography in combination with chemical ionization mass spectrometry. Deuterium was found to be incorporated into newly formed glycerol and newly synthesized palmitic and stearic acids (4-22% replacement) which were distributed throughout the molecular species in proportion to their relative rates of turnover (10-45% replacement). Within each unsaturation class, the palmitoyl species were labelled more extensively than the corresponding stearoyl species. Following in vitro incubation of the membranes with 10 mM Ca2+ at pH 8.5, there occurred a rapid degradation of the phosphatidylethanolamines (to 60% of control values after 90 min), while the phosphatidylcholines remained essentially unaffected. The various molecular species of the phosphatidylethanolamines were degraded linearly and in proportion to their masses. The deuterium content of the phosphatidylethanolamine and phosphatidylcholine remained constant throughout the incubation. It was concluded that under the present experimental conditions all molecular species of phosphatidylethanolamine, both old and newly synthesized, are equally accessible to the endogenous phospholipase.

A mechanism for epoxidation of cholesterol by hepatic microsomal lipid hydroperoxides

Evidence was obtained, using cis-stilbene as a model substrate, for the participation of peroxy and/or oxy radicals in epoxidation of cholesterol by rat liver microsomal phospholipid hydroperoxides and a ferrous ion-ADP complex. Under the conditions used, cholesterol was epoxidised to the α- and β-epoxides in the ratio 1:2–4, and cis-stilbene to trans-stilbene oxide without concomitant formation of the cis-oxide. Microsomal phospholipid hydroperoxides could be replaced with methyllinoleate monohydroperoxide for the epoxidation of both substrates. The hydroperoxide-mediated epoxidations were completely inhibited by oi-tocopherol and t-butylhydroxyanisole. A GLC study suggested that highly polyunsaturated fatty acyl constituents of the microsomal phospholipids might play an important role in epoxidation of the olefinic substrates.

Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2+ in rat liver microsomes.

1. Methods using t.l.c. and high-pressure liquid chromatography (h.p.l.c.) have been used to separate the complex variety of substances possessing a carbonyl function that are produced during lipid peroxidation. 2. The major type of lipid peroxidation studied was the ADP-Fe2+-stimulated peroxidation of rat liver microsomal phospholipids. Preliminary separation of the polar and non-polar products was achieved by t.l.c.: further separation and identification of individual components was performed by h.p.l.c. Estimations were performed on microsomal pellets and the supernatant mixture after incubation of microsomes for 30 min at 37 degrees C. 3. The polar fraction was larger than the non-polar fraction when expressed as nmol of carbonyl groups/g of liver. In the non-polar supernatant fraction the major contributors were n-alkanals (31% of the total), alpha-dicarbonyl compounds (22%) and 4-hydroxyalkenals (37%) with the extraction method used. 4. Major individual contributors to the non-polar fraction were found to be propanal, 4-hydroxynonenal, hexanal and oct-2-enal. Other components identified include butanal, pent-2-enal, hex-2-enal, hept-2-enal, 4-hydroxyoctenal and 4-hydroxyundecenal. The polar carbonyl fraction was less complex than the non-polar fraction, although the identities of the individual components have not yet been established. 5. Since these carbonyl compounds do not react significantly in the thiobarbituric acid reaction, which largely demonstrates the presence of malonaldehyde, it is concluded that considerable amounts of biologically reactive carbonyl derivatives are released in lipid peroxidation and yet may not be picked up by the thiobarbituric acid reaction.

Effects of cysteine and cystamine on the carbon tetrachloride induced decrease in arachidonic acid content of rat liver microsomal phospholipids

Cystamine and cysteine inhibited the carbon tetrachloride (CCl 4) prooxidant effect on rat liver microsomal preparations, at concentrations ranging from 0.001 mM to 1 mM. Cysteine exhibited a biphasic effect being an inhibitor of the prooxidant action at concentrations below 0.1 mM and acting as an enhancer at 1 mM. Cystamine but not cysteine pretreatment of the rats prevented the CCl 4 induced decrease in the arachidonic acid content of liver microsomal phospholipids. However, both cystamine and cysteine led to decreases in arachidonic acid similar to that produced by CCl 4 but they do not have deleterious effects on the liver. These results cast doubt on the role of lipid peroxidation in the liver cell injury by CCl 4.

Hydrolysis of membrane phospholipids by phospholipases of rat liver lysosomes.

(1) The hydrolysis of (32)P- or myo-[2-(3)H]inositol-labelled rat liver microsomal phospholipids by rat liver lysosomal enzymes has been studied. (2) The relative rates of hydrolysis of phospholipids at pH4.5 are: sphingomyelin>phosphatidylethanolamine>phosphatidylcholine> phosphatidylinositol. (3) The predominant products of phosphatidylcholine and phosphatidylethanolamine hydrolysis are their corresponding lyso-compounds, indicating a slow rate of total deacylation. (4) Ca(2+) inhibits the hydrolysis of all phospholipids, though only appreciably at high (>5mm) concentration. The hydrolysis of sphingomyelin is considerably less sensitive to Ca(2+) than that of glycerophospholipids. (5) Analysis of the water-soluble products of phosphatidylinositol hydrolysis (by using myo-[(3)H]inositol-labelled microsomal fraction as a substrate) produced evidence that more than 95% of the product is phosphoinositol, which was derived by direct cleavage from phosphatidylinositol, rather than by hydrolysis of glycerophosphoinositol. (6) This production of phosphoinositol, allied with negligible lysophosphatidylinositol formation and a detectable accumulation of diacylglycerol, indicates that lysosomes hydrolyse membrane phosphatidylinositol almost exclusively in a phospholipase C-like manner. (7) Comparisons are drawn between the hydrolysis by lysosomal enzymes of membrane substrates and that of pure phospholipid substrates, and also the possible role of phosphatidylinositol-specific lysosomal phospholipase C in cellular phosphatidylinositol catabolism is discussed.

Extensive exchange of rat liver microsomal phospholipids

Liver microsomal fractions were prepared from rats injected with a single dose of choline [ 14C] methylchloride or with single or multiple doses of 32P i. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85–95% exchangeable in 1–2 h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer.

Comparative studies of the responses of rat liver microsomal glucose-6-phosphatase and inorganic pyrophosphate-glucose phosphotransferase to phospholipase C treatment and phospholipid supplementation

1. 1.|Comparative studies of the responses of Glc-6- P phosphohydrolase and PP i-glucose phosphotransferase activities of rat liver microsomal glucose-6-phosphatase (EC 3.1.3.9) to in vitro phospholipid modifications have been carried out. Various conditions for assay enzymic activities were employed, and results compared. 2. 2.|Based on activity assays performed at pH 6 in the absence of added detergent, both activities decreased progressively and in parallel fashion with increasing length of exposure of microsomes to the action of phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). These activities of such treated preparation were increased by the addition of micellular dispersions of supplemental phospholipid (rat liver microsomal phospholipid preparations or, in one study, various classes of purified phospholipids). These observations provide yet another piece of evidence in support of catalysis of these two enzymic activities by a single microsomal enzyme. 3. 3.|The degree of restoration of activities towards original control levels through phospholipid supplementation following phospholipase C exposure of microsomes was much less when based on activity values obtained in the presence of the detergent deoxycholate than in its absence. 4. 4.|Enzymic activities of phospholipid-supplemented, phospholipase C-treated preparations were quite insensitive to stimulation by deoxycholate, while those of unexposed preparations were highly stimulated by this same detergent. 5. 5.|The pH-activity profile for PP i-glucose phosphotransferase activity obtained after phospholipase C exposure and phospholipid supplementation resembled strongly that noted with detergent-activated, otherwise untreated microsomal preparations, displaying a maximum at pH 5.6 in comparison with that at pH 4.5 exhibited by untreated microsomal preparations. 6. 6.|These observations are interpreted to indicate that phospholipid supplementation following phospholipase C exposure of rat liver microsomes does not lead to the restoration of microsomal glucose-6-phosphatase in its original form. Further, it is concluded that, in view of these findings and other observations in the literature, a reconsideration of the whole subject of the involvement of phospholipids in glucose-6-phosphatase action appears to be in order.

Studies on Microsomal Phospholipids That Inhibit Gulonolactone Oxidase

Abstract Fractions of rat liver microsomal phospholipid that inhibit gulonolactone oxidase are described. The inhibition was shown to be complete and occurs rapidly after the reaction has proceeded at the control rate for 20 to 30 min. It could be totally reversed by the addition of tocopherol, Mn++, Co++, or ethylenediaminetetraacetate. The inhibitory activity of the phospholipid was lost by enzymic cleavage of the β-acyl fatty acid moiety or by hydrolysis of the bases associated with the phosphatidyl group. Diglycerides derived from the lipid retained significant inhibitory properties. The inhibition is apparently accompanied by alterations of unsaturated fatty acids resulting in formation of chromogenic substances that react with thiobarbituric acid. When the degree of unsaturation of the inhibitor phospholipid is reduced, both the inhibitory properties and formation of chromogens are proportionally decreased.