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European Journal of Biochemistry Mar 2004Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of...
Recent studies have suggested that parts of the hepatic activities of diacylglycerol acyltransferase and acyl cholesterol acyltransferase are expressed in the lumen of the endoplasmic reticulum (ER). However the ER membrane is impermeable to the long-chain fatty acyl-CoA substrates of these enzymes. Liver microsomal vesicles that were shown to be at least 95% impermeable to palmitoyl-CoA were used to demonstrate the membrane transport of palmitoylcarnitine and free L-carnitine - processes that are necessary for an indirect route of provision of ER luminal fatty acyl-CoA through a luminal carnitine acyltransferase (CAT). Experimental conditions and precautions were established to permit measurement of the transport of [14C]palmitoylcarnitine into microsomes through the use of the luminal CAT and acyl-CoA:ethanol acyltransferase as a reporter system to detect formation of luminal [14C]palmitoyl-CoA. Rapid, unidirectional transport of free L-[3H]carnitine by microsomes was measured directly. This process, mediated either by a channel or a carrier, was inhibited by mersalyl but not by N-ethylmaleimide or sulfobetaine - properties that differentiate it from the mitochondrial inner membrane carnitine/acylcarnitine exchange carrier. These findings are relevant to the understanding of processes for the reassembly of triacylglycerols that lipidate very low density lipoprotein particles as part of a hepatic triacylglycerol lipolysis/re-esterification cycle.
Topics: Animals; Biological Transport; Carnitine; Endoplasmic Reticulum; Enzyme Inhibitors; Genes, Reporter; Intracellular Membranes; Male; Mersalyl; Microsomes, Liver; Palmitoyl Coenzyme A; Palmitoylcarnitine; Rats; Rats, Sprague-Dawley
PubMed: 15009207
DOI: 10.1111/j.1432-1033.2004.03997.x -
Plant Physiology Mar 1999The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake...
The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4, 4'-dithiocyanostilbene-2,2'-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.
PubMed: 10069840
DOI: 10.1104/pp.119.3.1025 -
Biochimica Et Biophysica Acta Feb 1997The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined...
The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.
Topics: Animals; Arsenicals; Binding Sites; Calcium; Ethylmaleimide; In Vitro Techniques; Intracellular Membranes; Membrane Proteins; Mersalyl; Mitochondria, Liver; Mitochondrial Swelling; Permeability; Peroxides; Rats; Rats, Wistar; Sulfhydryl Compounds; Sulfhydryl Reagents; tert-Butylhydroperoxide
PubMed: 9048976
DOI: 10.1016/s0005-2728(96)00111-9 -
British Medical Journal Nov 1965
Clinical Trial Comparative Study Randomized Controlled Trial
Topics: Adult; Aged; Bendroflumethiazide; Diuretics; Edema; Ethacrynic Acid; Female; Furosemide; Heart Failure; Humans; Hypertension, Portal; Male; Middle Aged; Natriuresis; Nephrotic Syndrome; Organomercury Compounds
PubMed: 5849145
DOI: 10.1136/bmj.2.5473.1277 -
Biochimica Et Biophysica Acta Mar 1999Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2',...
Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used to determine total transport (carrier mediated and diffusion). Both lactate and pyruvate transport could be inhibited by a combination of 0.5 mM 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid, 5 mM mersalyl and 10 mM alpha-cyano-4-hydroxycinnamate. The kinetic parameters, Km and Vmax, for carrier-mediated transport of lactate were 9.9+/-1.1 mM and 0. 69+/-0.02 mmol l-1 s-1, respectively. For pyruvate, Km and Vmax were 4.4+/-1.3 mM and 0.30+/-0.05 mmol l-1 s-1, respectively. The diffusion component of the total transport was 0.0040+/-0.0005[S] (n=4) and 0.0048+/-0.0003[S] (n=4) for lactate and pyruvate, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes.
Topics: Animals; Biological Transport; Carrier Proteins; Cell Differentiation; Cells, Cultured; Creatine Kinase; Fluoresceins; Fluorescent Dyes; Kinetics; Lactic Acid; Monocarboxylic Acid Transporters; Muscle, Skeletal; Protein Isoforms; Pyruvic Acid; Rats; Rats, Wistar
PubMed: 10082802
DOI: 10.1016/s0005-2736(99)00009-7 -
European Journal of Biochemistry Nov 1975The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1-10 muM) of the mercurial diuretic mersalyl. Complete...
The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1-10 muM) of the mercurial diuretic mersalyl. Complete inhibition was obtained with 0.1 mM mersalyl. Similar effects were observed whether the adenylate cyclase preparation was assayed in the presence of 10 muM GTP, 0.1 muM glucagon, 10 mM NaF or without any addition. The effect of mersalyl was not due to inhibition of the regenerating system present in the incubation medium, since the effect of mersalyl was preserved and even enhanced in its absence. The inhibition brought about by mersalyl was due to both a decrease of the maximal velocity of the reaction and of the affinity of the enzyme for the substrate. It was immediate, and irreversible spontaneously, but it was reversed by the simultaneous additions of 2-mercaptoethanol, in a dose-dependent fashion. Other -SH reagents were found to have an effect equal to, or lower than, that of mersalyl. Mersalyl had no effect upon Mg2+-ATPase, although it inhibited the (Na+-K+) activated ATPase. Since mersalyl is known to be a 'non-penetrant' reagent, it is postulated that a catalytically important, mercurial-sensitive, part of adenylate cyclase is at the surface of the plasma membrane. This view is supported by the following facts: (a) mersalyl acted with a similar dose-response curve upon an intact as well as a detergent-dispersed cyclase preparation while no effect was observed upon a solubilized Mg2+-ATPase preparation; (b) a covalent p-chloromercuribenzoate-Sephadex preparation (but not its supernatant) inhibited the cyclase from intact membranes. It is proposed that mercurial derivatives, by their relative specificity of action (no effect on Mg2+-ATPase), can serve as useful probes in the elucidation of the multicomponent structure of the cyclase system.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Cell Membrane; Chloromercuribenzoates; Dose-Response Relationship, Drug; Enzyme Activation; Female; Fluorides; Glucagon; Guanosine Triphosphate; Kinetics; Liver; Magnesium; Mercaptoethanol; Mercury; Mersalyl; Organomercury Compounds; Organometallic Compounds; Rats
PubMed: 128456
DOI: 10.1111/j.1432-1033.1975.tb02487.x -
European Journal of Biochemistry Aug 2000Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake...
Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.
Topics: Animals; Atractyloside; Chromatography, Gel; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavin-Adenine Dinucleotide; Glutamate Dehydrogenase; Kinetics; L-Lactate Dehydrogenase; Male; Mitochondria, Liver; Models, Biological; Nucleotidyltransferases; Phosphotransferases (Alcohol Group Acceptor); Rats; Rats, Wistar; Riboflavin; Time Factors
PubMed: 10903524
DOI: 10.1046/j.1432-1327.2000.01552.x -
Plant Physiology Jul 1983When assayed in the presence of azide, NO(3) (-) was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 x M017) root...
When assayed in the presence of azide, NO(3) (-) was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 x M017) root microsomal membranes. The distribution of the NO(3) (-)-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H(+)-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 mum vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO(3) (-)-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H(+) from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K(+) > NH(4) (+) > Rb(+) > Cs(+) > Li(+) > Na(+) > choline(+)) whereas the NO(3) (-)-sensitive ATPase was stimulated by anions (Cl(-) > Br(-) > C(2)H(3)O(2) (-) > SO(4) (2-) > I(-) > HCO(3) (-) > SCN(-)). Both ATPases required divalent cations. However, the order of preference for the NO(3) (-)-sensitive ATPase (Mn(2+) > Mg(2+) > Co(2+) > Ca(2+) > Zn(2+)) differed from that of the vanadate-sensitive ATPase (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO(3) (-)-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO(3) (-)-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO(3) (-)-sensitive ATPase was stimulated by gramicidin and was associated with NO(3) (-)-inhibitable H(+) transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO(3) (-)-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO(3) (-) sensitivity is a better criterion by which to identify this ATPase than either Cl(-) stimulation or H(+) transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.
PubMed: 16663096
DOI: 10.1104/pp.72.3.837 -
Canadian Medical Association Journal Aug 1956
Topics: Diuretics; Drug Overdose; Mersalyl; Organomercury Compounds; Poisoning
PubMed: 13343095
DOI: No ID Found -
The Biochemical Journal Feb 1976Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant...
Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.
Topics: Animals; Biological Transport; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carboxylic Acids; Carcinoma, Ehrlich Tumor; Cinnamates; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Lactates; Mersalyl; Mice; Nigericin; Protons; Temperature; Valinomycin
PubMed: 7237
DOI: 10.1042/bj1540405