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Rapeseed oil-rich diet alters in vitro menadione and nimesulide hepatic mitochondrial toxicity

Monteiro, João P.; Silva, Ana M.; Jurado, A. S.; Oliveira, Paulo J.
Fonte: Elsevier Ltd. Publicador: Elsevier Ltd.
Tipo: Artigo de Revista Científica
ENG
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Diet-induced changes in the lipid composition of mitochondrial membranes have been shown to influence physiological processes. However, the modulation effect of diet on mitochondrially-active drugs has not yet received the deserved attention. Our hypothesis is that modulation of membrane dynamics by diet impacts drug-effects on liver mitochondrial functioning. In a previous work, we have shown that a diet rich in rapeseed oil altered mitochondrial membrane composition and bioenergetics in Wistar rats. In the present work, we investigated the influence of the modified diet on hepatic mitochondrial activity of two drugs, menadione and nimesulide, and FCCP, a classic protonophore, was used for comparison. The results showed that the effects of menadione and nimesulide were less severe on liver mitochondria for rats fed the modified diet than on rats fed the control diet. A specific effect on complex I seemed to be involved in drug-induced mitochondria dysfunction. Liver mitochondria from the modified diet group were more susceptible to nimesulide effects on MPT induction. The present work demonstrates that diet manipulation aimed at modifying mitochondrial membrane properties alters the toxicity of mitochondria active agents. This work highlights that diet may potentiate mitochondrial pharmacologic effects or increase drug-induced liabilities.; The project was supported by the Foundation for Science and Technology (FCT...

Rearrangement of the Chloroplast Thylakoid at Chilling Temperature in the Light 1

Mäenpää, Pirkko; Aro, Eva-Mari; Somersalo, Susanne; Tyystjärvi, Esa
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1988 EN
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The leaves of chilling-sensitive pumpkin (Cucurbita pepo L.) showed symptoms reminiscent of photoinhibition when kept for 4 days at 5°C in moderate light. A decrease was observed in the variable part of chlorophyll α fluorescence, apparent quantum yield, and maximum rate of O2 evolution. Chloroplast whole-chain electron transport activity measured from chloroplast thylakoids had decreased to 51% of the control value. Photosystem II (PSII) activity decreased by only 9%, suggesting that photoinhibition was not responsible for the loss of electron transport activity. An increase in the proportion of PSIIβ (measured as a βmax value) was observed after the chilling treatment. Fractionation of thylakoid membranes showed a 42% increase in PSII activity in the nonappressed region while that in the appressed region decreased slightly. This was accompanied by a decrease in the ratio of the length of appressed to nonappressed thylakoid membranes. Leaf photosynthesis largely recovered within 24 hours of returning to the original growth conditions. We suggest that the increase in the proportion of PSIIβ during chilling in light plays a role in protecting PSII from photoinhibitory damage.

Potassium Channels in Motor Cells of Samanea saman1: A Patch-Clamp Study

Moran, Nava; Ehrenstein, Gerald; Iwasa, Kunihiko; Mischke, Charles; Bare, Charles; Satter, Ruth L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1988 EN
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Leaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K+, Cl− and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K+ currents that flow during leaflet movement. Recordings in the “whole-cell” mode reveal depolarization-activated K+ currents in extensor and flexor cells that increase slowly (t½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K+ currents is adequate to account quantitatively for K+ loss, previously measured in vivo during cell shrinkage. The K+ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light- and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity 1

Peeler, Thomas C.; Stephenson, Martha B.; Einspahr, Kregg J.; Thompson, Guy A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1989 EN
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We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.

Changes in the Physical State of Membrane Lipids during Senescence of Rose Petals 1

Faragher, John D.; Wachtel, Ellen; Mayak, Shimon
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1987 EN
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479.00406%
Changes in the physical state of microsomal membrane lipids during senescence of rose flower petals (Rosa hyb. L. cv Mercedes) were measured by x-ray diffraction analysis. During senescence of cut flowers held at 22°C, lipid in the ordered, gel phase appeared in the otherwise disordered, liquid-crystalline phase lipids of the membranes. This was due to an increase in the phase transition temperature of the lipids. The proportion of gel phase in the membrane lipids of 2-day-old flowers was estimated as about 20% at 22°C. Ethylene may be responsible, at least in part, for the increase in lipid transition temperature during senescence since aminooxyacetic acid and silver thiosulfate inhibited the rise in transition temperature. When flowers were stored at 3°C for 10 to 17 days and then transferrd to 22°C, gel phase lipid appeared in membranes earlier than in freshly cut flowers. This advanced senescence was the result of aging at 3°C, indicated by increases in membrane lipid transition temperature and ethylene production rate during the time at 3°C. It is concluded that changes in the physical state of membrane lipids are an integral part of senescence of rose petals, that they are caused, at least in part, by ethylene action and that they are responsible...

Enhanced Thermal Tolerance in a Mutant of Arabidopsis Deficient in Palmitic Acid Unsaturation 1

Kunst, Ljerka; Browse, John; Somerville, Chris
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1989 EN
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478.51688%
A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast ω9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO2 fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28°C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.

Accumulation of Chlorophyll a/b-Binding Polypeptides in Chlamydomonas reinhardtii y-1 in the Light or Dark at 38°C 1: Evidence for Proteolytic Control

Hoober, J. Kenneth; Maloney, Margaret A.; Asbury, Linda R.; Marks, Dawn B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1990 EN
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The kinetics of accumulation of light harvesting chlorophyll (Chl) a/b-binding polypeptides (LHCPs) in thylakoid membranes were analyzed during greening of Chlamydomonas reinhardtii y-1 at 38°C. Initial accumulation of LHCPs in thylakoid membranes was linear; LHCP precursors or polypeptides in transit within the chloroplast stroma were not detected. The rate of accumulation in the light was at least five-fold greater than that in the dark. The relatively small amount of LHCPs that accumulated in the dark was integrated properly in the membrane, as judged by the pattern of cleavage in vitro by exogenous proteases, and did not turn over at a significant rate in vivo. The kinetic data suggested that in y-1 cells either translation of LHCP mRNA was inhibited in the dark or newly synthesized polypeptides were degraded concurrently with transport into the chloroplast unless rescued by Chl. LHCPs accumulated in cells of the Chl b-deficient strain pg-113 at the same rate in the dark or the light at 38°C, an indication that light did not affect translation of LHCP mRNA. Membrane-associated LHCPs in pg-113 cells were completely degraded, in contrast to those in y-1 cells, by exogenous proteases, which suggested that pg-113 cells are deficient in a proteolytic activity. A peptidase was recovered from y-1 cells in a membrane fraction with a buoyant density slightly less than that of thylakoid membranes. Although a role for this activity in degradation of LHCPs has not been established...

Common Identity of Substrate Binding Subunit of Vacuolar H+-Translocating Inorganic Pyrophosphatase of Higher Plant Cells 1

Rea, Philip A.; Britten, Christopher J.; Sarafian, Vahe
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1992 EN
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There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H+-translocating inorganic pyrophosphatase (tonoplast H+-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (Mr) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H+-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H+-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (Mr(Beta) = 64,500; Mr(Vigna) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [14C]-N-ethylmaleimide. Similarly, the Mr 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the Mr 66...

Tonoplast Vesicles of Opposite Sidedness from Soybean Hypocotyls by Preparative Free-Flow Electrophoresis 1

Canut, Hervé; Brightman, Andrew; Boudet, Alain M.; Morré, D. James
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1990 EN
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474.6798%
Tonoplast vesicles were purified from a microsomal fraction isolated from etiolated soybean hypocotyls (Glycine max L.) by preparative free-flow electrophoresis. Marker enzyme determinations and immunoblot analysis against the vacuolar-ATPase confirmed the nature and the purity of the isolated membranes. A purified tonoplast fraction also was obtained by consecutive sucrose and glycerol centrifugation which was further resolved into two different populations of vesicles (TA and TB) by free-flow electrophoresis. The determination of the sidedness of these different vesicles included concanavalin A binding as an imposed label, NADH-ferricyanide oxidoreductase cytochemistry, and ATPase latency. The tonoplast fractions, obtained by consecutive sucrose and glycerol gradient centrifugations, were found to consist of a mixture of two populations of vesicles of opposite sidedness. The least electronegative fraction obtained by free-flow electrophoresis (TB) consisted predominantly of cytoplasmic side out tonoplast vesicles while a fraction of greater electronegativity (TA) contained the cytoplasmic side in tonoplast vesicles. The relative amounts of each type of vesicle varied with the method of homogenization. Razor blade chopping, Polytron...

Characterization of the Major Integral Protein of Vacuolar Membrane 1

Maeshima, Masayoshi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1992 EN
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481.71527%
The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N′-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H+-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.

Multiple Forms of Plant Cytochromes P-450 1

Donaldson, Robert P.; Luster, Douglas G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1991 EN
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474.78176%
Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases are implicated in the metabolism of sterols, terpenes, gibberellins, isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 are involved in the detoxification of herbicides (chlorotoluron, primsulfuron, and diclofop) includes photoreversible CO inhibition of the reactions, and a requirement for O2 and NADPH. Several cytochromes P-450, Mr 45,000 to 65,000, have been isolated, including hydroxylases of cinnamic acid, 3,9-dihydroxypterocarpan, and digitoxin. In some cases the purified cytochrome P-450 has been successfully reconstituted with NADPH:cytochrome P-450 reductase (Mr 72,000-84,000 protein). This reductase appears to be a nonspecific electron donor to different forms of cytochrome P-450. Immunological techniques and specific inhibitors (triazoles, imidazole derivatives) are being used to characterize plant cytochromes P-450 and the NADPH:cytochrome P-450 reductase. Specific cytochromes P-450 are induced by wounding or pathogens, others are expressed in specific cell types. Plant cytochromes P-450 are found in various subcellular locations, including endoplasmic reticulum, plasma membranes, glyoxysomes...

Effects of Temperature on the Coupled Activities of the Vanadate-Sensitive Proton Pump from Maize Root Microsomes

Brauer, David; Loper, Matthew; Schubert, Carol; Tu, Shu-I
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1991 EN
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The mechanism by which proton transport is coupled to ATP hydrolysis by vanadate-sensitive pumps is poorly understood. The effects of temperature on the activities of the vanadate-sensitive ATPase from maize (Zea mays) roots were assessed to provide insight into the coupling mechanism. The initial rate of proton transport had a bell-shaped dependence on temperature with an optimal range between 20 and 30°C. However, the rate of vanadate-sensitive ATP hydrolysis increased as the temperature was raised from 4 to 43°C. The differential sensitivity of proton transport to temperatures above 30°C was also observed when the ATPase was reconstituted into dioleoylphosphatidylcholine vesicles. Inhibition of proton transport with temperatures above 30°C was associated with higher rates of proton leakage from the membranes. In addition, proton transport was more inhibited than ATP hydrolysis at temperatures below 10°C. Reduced rates of proton transport at lower temperatures were not associated with higher rate of proton conductivity across the membranes. Therefore, the preferential inhibition of proton transport at temperatures below 10°C may reflect an effect of temperature on the coupling between proton transport and ATP hydrolysis within the vanadate-sensitive ATPase.

Differentiation between Several Types of Phosphohydrolases in Light Microsomes of Corn Roots 1

Vicente, Joaquim A.F.; Vale, M. Graça P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1991 EN
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479.5072%
The phosphohydrolase activity of a light microsomal fraction isolated from corn roots (Zea mays L. cv LG 55) was investigated. The fraction, which appears to be enriched in endoplasmic reticulum and Golgi membranes, has ATPase and pyrophosphatase activities that hydrolyze ATP and pyrophosphate at an optimum pH of 7.0, with Km values of about 160 and 240 micromolar and with Vmax values of about 200 and 50 nanomoles substrate hydrolyzed per milligram protein per minute, respectively. These enzymes differ in their sensitivity to anions and inhibitors. The ATPase is stimulated by sulfate anions, whereas pyrophosphatase is inhibited by molybdate. Furthermore, the simultaneous addition of ATP and pyrophosphate to the reaction medium increases phosphohydrolysis, suggesting that separate enzymes are operating in the membranes. We also observed that pyrophosphate competitively inhibits the ATPase, whereas ATP has no significant effect on the pyrophosphatase. On the other hand, we observed a detergent-stimulated, molybdate-insensitive inosine diphosphatase activity which, in the native state, hydrolyzes inosine diphosphate with a Km of about 700 micromolar and a Vmax of about 450 nanomoles inosine diphosphate hydrolyzed per milligram protein per minute. In the solubilized form...

Immunological Characterization of Two Dominant Tonoplast Polypeptides 1

Betz, Marion; Dietz, Karl-Josef
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1991 EN
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At least 14 distinct polypeptides reside in the tonoplast of barley (Hordeum vulgare) mesophyll vacuoles. Two of the polypeptides were isolated from two-dimensional separations of vacuoplast membrane proteins and used for immunization. With the antisera, the localization on the membrane and the distribution of the polypeptides in the plant kingdom and in various tissues of barley plants was studied. The polypeptides have an apparent molecular mass of 31 and 40 kilodaltons. After freeze-thaw cycles or washing of the membranes with 4.5 millimolar NaCl, the polypeptides were still sedimented with the membranes, suggesting an intrinsic localization. The antiserum against the 31-kilodalton polypeptide bound to the outer surface of isolated intact vacuoles. In chromatographic separations of Triton X-100-solubilized membrane fractions, the residual activities of various acid hydrolases eluted distinct from the 31- and 40-kilodalton polypeptides. Both polypeptides tend to form larger aggregates, however smaller than the tonoplast ATPase. Cross-reactive polypeptides were present in higher and lower plants (the green alga Chara corallina and the liverwort Conocephalum) and in liver tissue from rat and beef, but were not detected in other animal tissues tested so far. The results indicate a wide distribution of these tonoplast polypeptides in vacuole-containing organisms.

The orientation of iron–sulphur clusters in membrane multilayers prepared from aerobically-grown Escherichia coli K12 and a cytochrome-deficient mutant

Blum, Haywood; Poole, Robert K.; Ohnishi, Tomoko
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/08/1980 EN
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1. Membrane particles prepared from ultrasonically-disrupted, aerobically-grown Escherichia coli were centrifuged on to a plastic film that was supported perpendicular to the centrifugal field to yield oriented membrane multilayers. In such preparations, there is a high degree of orientation of the planes of the membranes such that they lie parallel to each other and to the supporting film. 2. When dithionite- or succinate-reduced multilayers are rotated in the magnetic field of an e.p.r. spectrometer, about an axis lying in the membrane plane, angular-dependent signals from an iron–sulphur cluster at gx=1.92, gy=1.93 and gz=2.02 are seen. The g=1.93 signal has maximal amplitude when the plane of the multilayer is perpendicular to the magnetic field. Conversely, the g=2.02 signal is maximal when the plane of the multilayer is parallel with the magnetic field. 3. Computer simulations of the experimental data show that the cluster lies in the cytoplasmic membrane with the gy axis perpendicular to the membrane plane and with the gx and gz axes lying in the membrane plane. 4. In partially-oxidized multilayers, a signal resembling the mitochondrial high-potential iron–sulphur protein (Hipip) is seen whose gz=2.02 axis may be deduced as lying perpendicular to the membrane plane. 5. Appropriate choice of sample temperature and receiver gain reveals two further signals in partially-reduced multilayers: a g=2.09 signal arises from a cluster with its gz axis in the membrane plane...

The incorporation of radioactive fatty acids into the phospholipids of nerve-cell-body membranes in vivo. Evidence for highly labelled neuronal nuclei

Baker, R. Roy; Chang, Huu-yi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/04/1980 EN
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479.5072%
1. Nerve cell bodies were isolated in bulk from cerebral cortices of 15 day-old rabbits after intrathecal injections of [3H]plamitate, [3H]oleate or [3H]arachidonate and [14C]glycerol. 2. Nuclear, microsomal and two mitochondrial fractions were isolated from homogenates of the radioactively labelled nerve cell bodies by using differential and discontinuous-gradient centrifugation. 3. After 7.5min in vivo, a high percentage (>80%) of the total 3H-labelled fatty acid radioactivity was found in the membrane fractions of the nerve cell bodies, whereas after 60min in vivo 50% of the total [14C]glycerol radioactivity was found in the high-speed supernatant. 4. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, and the radioactivity in neutral lipid and non-esterified fatty acid fractions were determined in the four subfractions, as were the distributions of several marker enzymes and nucleates. 5. With respect of 3H-labelled fatty acid, the phospholipids of the nuclear fraction had the highest specific radioactivities of the four subfractions. However, for [14C]glycerol labelling, generally the 14C specific radioactivities for individual phospholipids were comparable in the four subfractions. This latter observation suggests transport of phospholipids synthesized de novo between membranes of the nerve cell body. 6. Double-labelling experiments demonstrated that individual phospholipids and the combined neutral lipids of the nuclear fraction had higher labelling ratios of 3H-labelled fatty acid/[14C]glycerol than did the corresponding lipids of the microsomal or mitochondrial fractions. 7. On the basis of the labelling results and the marker studies...

AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase*

Chen, Yan Qun; Kuo, Ming-Shang; Li, Shuyu; Bui, Hai H.; Peake, David A.; Sanders, Philip E.; Thibodeaux, Stefan J.; Chu, Shaoyou; Qian, Yue-Wei; Zhao, Yang; Bredt, David S.; Moller, David E.; Konrad, Robert J.; Beigneux, Anne P.; Young, Stephen G.; Cao, G
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 11/04/2008 EN
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AGPAT6 is a member of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family that appears to be important in triglyceride biosynthesis in several tissues, but the precise biochemical function of the enzyme is unknown. In the current study, we show that AGPAT6 is a microsomal glycerol-3-phosphate acyltransferase (GPAT). Membranes from HEK293 cells overexpressing human AGPAT6 had higher levels of GPAT activity. Substrate specificity studies suggested that AGPAT6 was active against both saturated and unsaturated long-chain fatty acyl-CoAs. Both glycerol 3-phosphate and fatty acyl-CoA increased the GPAT activity, and the activity was sensitive to N-ethylmaleimide, a sulfhydryl-modifying reagent. Purified AGPAT6 protein possessed GPAT activity but not AGPAT activity. Using [13C7]oleic acid labeling and mass spectrometry, we found that overexpression of AGPAT6 increased both lysophosphatidic acid and phosphatidic acid levels in cells. In these studies, total triglyceride and phosphatidylcholine levels were not significantly altered, although there were significant changes in the abundance of specific phosphatidylcholine species. Human AGPAT6 is localized to endoplasmic reticulum and is broadly distributed in tissues. Membranes of mammary epithelial cells from Agpat6-deficient mice exhibited markedly reduced GPAT activity compared with membranes from wild-type mice. Reducing AGPAT6 expression in HEK293 cells through small interfering RNA knockdown suggested that AGPAT6 significantly contributed to HEK293 cellular GPAT activity. Our data indicate that AGPAT6 is a microsomal GPAT...

Reduction of Clofazimine by Mycobacterial Type 2 NADH:Quinone Oxidoreductase: A PATHWAY FOR THE GENERATION OF BACTERICIDAL LEVELS OF REACTIVE OXYGEN SPECIES*

Yano, Takahiro; Kassovska-Bratinova, Sacha; Teh, J. Shin; Winkler, Jeffrey; Sullivan, Kevin; Isaacs, Andre; Schechter, Norman M.; Rubin, Harvey
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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The mechanism of action of clofazimine (CFZ), an antimycobacterial drug with a long history, is not well understood. The present study describes a redox cycling pathway that involves the enzymatic reduction of CFZ by NDH-2, the primary respiratory chain NADH:quinone oxidoreductase of mycobacteria and nonenzymatic oxidation of reduced CFZ by O2 yielding CFZ and reactive oxygen species (ROS). This pathway was demonstrated using isolated membranes and purified recombinant NDH-2. The reduction and oxidation of CFZ was measured spectrally, and the production of ROS was measured using a coupled assay system with Amplex Red. Supporting the ROS-based killing mechanism, bacteria grown in the presence of antioxidants are more resistant to CFZ. CFZ-mediated increase in NADH oxidation and ROS production were not observed in membranes from three different Gram-negative bacteria but was observed in Staphylococcus aureus and Saccharomyces cerevisiae, which is consistent with the known antimicrobial specificity of CFZ. A more soluble analog of CFZ, KS6, was synthesized and was shown to have the same activities as CFZ. These studies describe a pathway for a continuous and high rate of reactive oxygen species production in Mycobacterium smegmatis treated with CFZ and a CFZ analog as well as evidence that cell death produced by these agents are related to the production of these radical species.

Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin-granule `ghosts'

Apps, David K.; Pryde, James G.; Sutton, Raul; Phillips, John H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/08/1980 EN
Relevância na Pesquisa
479.58043%
1. Highly purified resealed chromaffin-granule `ghosts' were assayed for ATPase and ATP-driven H+-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H+-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H+-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the `ghosts' with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H+-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded `ghosts', whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with 14C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[14C]carbodi-imide are labelled predominantly in a protein of low molecular weight...

Effect of growth conditions on the involvement of cytochrome c in electron transport, proton translocation and ATP synthesis in the facultative methylotroph Pseudomonas AM1

Keevil, C. William; Anthony, Christopher
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/07/1979 EN
Relevância na Pesquisa
474.91016%
The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O2 have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. →H+/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the →H+/O ratio increased to about 6. Cell yields with respect to succinate and O2 were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH4+-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the `needs' of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a3. In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a3 occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a3...