PubMed Health⌕ Search

Biomedical subjects

E Rial

Publications and source records attributed to E Rial.

At least 37 records · Page 2Linked to original sources
PubMed1990

Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria.

The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.

Adipose Tissue, Brown↗
PubMed1989

Effect of hydrophobic sulphydryl reagents on the uncoupling protein and inner-membrane anion channel of brown-adipose-tissue mitochondria.

The effects of three sulphydryl reagents of differing hydrophobicity (N-ethylmaleimide, N-benzylmaleimide and N,N'-o-phenylenedimaleimide) on ion permeation through the inner membrane of brown-adipose-tissue mitochondria are investigated. GDP-sensitive permeation of chloride and protons (hydroxyl ions) through the uncoupling protein is increased exponentially with time by all three reagents. With increasing hydrophobicity of the reagents, modification is enhanced and an initial inhibited state becomes apparent. Results are interpreted in terms of a two-stage modification via a non-transporting intermediate, which does not bind GDP, to a final highly conducting product. The reagents also react with a hydrophilic sulphydryl group on an independent protein to induce a GDP-insensitive pathway which allows chloride, phosphate and sulphate to cross the membrane. The use of different sulphydryl reagents allows the two pathways to be clearly distinguished.

Adipose Tissue, Brown↗
PubMed1987

The uncoupling protein from brown-adipose-tissue mitochondria. Chymotrypsin-induced structural and functional modifications.

Mitoplasts prepared from brown adipose tissue mitochondria were treated with chymotrypsin and the fragments derived from the 32-kDa uncoupling protein identified by immunoblotting. Extensive proteolysis of the uncoupling protein occurred, the polypeptide pattern being affected by binding of the inhibitory nucleotide GDP. Chymotrypsin modifies the nucleotide binding site, lowering its affinity from 1.7 microM to 21 microM but without decreasing its binding capacity. Nucleotide bound to the modified site can still inhibit the permeation of H+ and Cl- through the protein. The ion conducting pathway itself is also sensitive to chymotrypsin, Cl- and H+ transport being partially inhibited in parallel. The ability of fatty acids to increase the H+ permeability of the protein is also inhibited in parallel with the basal H+ permeability. The results confirm that the transport of H+ and Cl-, and the fatty acid regulation of H+ permeation all share a common structural element within the 32-kDa protein.

Adipose Tissue, Brown↗
PubMed1986

Chemical modification of the brown-fat-mitochondrial uncoupling protein with tetranitromethane and N-ethylmaleimide. A cysteine residue is implicated in the nucleotide regulation of anion permeability.

Treatment of brown adipose tissue mitochondria with tetranitromethane or N-ethylmaleimide decreases the affinity with which inhibitory nucleotide GDP binds to the tissue-specific uncoupling protein. Both reagents modify cysteine residues which are 'accessible' and 'buried' to 5,5'-dithio-bis(2-nitrobenzoic acid) (Nbs2). Modification of the single Nbs2-accessible residue correlates with the loss of high-affinity binding sites for GDP. Tetranitromethane does not affect the Cl- or H+ permeability of the protein in the absence of nucleotide, while N-ethylmaleimide increases both by 70-80%. Bound GDP is a less effective inhibitor of Cl- permeability after N-ethylmaleimide or tetranitromethane treatment, but retains much of the ability to inhibit H+ permeation.

Adipose Tissue, Brown↗
PubMed1985

The characterization and energetic potential of brown adipose tissue in man.

In adult man, brown fat can be detected in perinephric fat depots by visual inspection, electron microscopy and nucleotide binding to the tissue-specific uncoupling protein. The 32 kDa uncoupling protein is functionally active, showing a nucleotide-sensitive conductance to protons and an uncoupling response to fatty acids. The amount of uncoupling protein in human mitochondria is equivalent to that in a partially cold-adapted guinea pig, indicating some potential for thermogenesis. Respiratory capacity measurements indicate that the total perinephric fat in adult man can only account for one-fivehundredth of the whole-body response to infused noradrenaline. Thus, although brown fat has been found to be quantitatively important in animal studies, considerable caution must be exercised in extrapolating its significance to adult man.

Adipose Tissue, Brown↗
PubMed1984

The mitochondrial uncoupling protein from guinea-pig brown adipose tissue. Synchronous increase in structural and functional parameters during cold-adaptation.

The time-course for the induction of the uncoupling pathway in the inner membrane of brown-fat mitochondria from cold-adapting guinea pigs was studied. The amount of the protein was quantified from the capacity for high-affinity binding of GDP to the intact mitochondria, and was compared with the functional parameters diagnostic of the protein, namely the nucleotide-sensitive proton conductance and the sensitivity to uncoupling by low concentrations of fatty acids. A monophasic increase in nucleotide titre was observed, with no evidence of an early 'unmasking' of preexisting nucleotide-binding sites. The nucleotide-sensitive conductance increased in precise synchrony with the nucleotide-binding capacity. Mitochondria from newborn animals, and those from acutely cold-adapted animals, showed anomalously low sensitivities to uncoupling by fatty acids.

Adaptation, Physiological↗
PubMed1984

Physiological state of submitochondrial particles and their susceptibility to Triton X-100.

The solubilizing effect of Triton X-100 on beef heart submitochondrial particles (ETPH) has been studied under various physiological conditions. Coupled, uncoupled and azide-inhibited ETPH particles have been studied. Quantitative and qualitative differences are found in the proteins solubilized by the detergent from ETPH particles under the various conditions tested.

Animals↗
PubMed1984

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes.

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.

Animals↗
PubMed1983

Brown-adipose-tissue mitochondria: the regulation of the 32000-Mr uncoupling protein by fatty acids and purine nucleotides.

The increased proton permeability induced by the addition of a synthetic proton translocator to non-respiring hamster brown-fat mitochondria is unaffected by purine nucleotide addition. In contrast the permeability induced by fatty acids is inhibited by nucleotide, indicating that fatty acids act at the 32000-Mr uncoupling protein. Fatty acids lower the affinity of nucleotide binding to the 32000-Mr protein, but not sufficiently to explain their uncoupling action. The sensitivity of the fatty acid modulation of permeability is dependent on chain length, extent of unsaturation and pH. There is a requirement for an unesterified carboxyl group. In respiring mitochondria fatty acids act in the presence of nucleotide by lowering the 'break-point' potential at which the conductance of the 32000-Mr protein increases. Fatty acids have no effect on the chloride uniport activity of the 32000-Mr protein, but decouple the interference between chloride and protons when the simultaneous transport of both ions is attempted.

Adipose Tissue, Brown↗
PubMed1983

The regulation of the proton conductance of brown fat mitochondria. Identification of functional and non-functional nucleotide-binding sites.

The binding of purine nucleotides to intact brown fat mitochondria is re-examined. In addition to the previously reported high affinity binding site, a low-affinity site is found, which requires several minutes to saturate. Only the high affinity site is functional in regulating the proton and halide permeabilities of the mitochondria. The low affinity site can introduce errors in the use of nucleotide binding to quantitate the Mr 32000 uncoupling protein unique to these mitochondria.

Adipose Tissue, Brown↗
PubMed1982

Fatty acids as acute regulators of the proton conductance of hamster brown-fat mitochondria.

Possible mechanisms are evaluated for the acute regulation of the hamster brown-fat mitochondrial proton-conductance pathway which is active during non-shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non-thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl-CoA and acylcarnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl-CoA potentiates the conductance increase. An effect of acyl-CoA upon chloride permeability is not specific to brown-fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl-CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown-fat-specific 32000-Mr protein is discussed.

Adipose Tissue, Brown↗
PubMed1982

The acute regulation of mitochondrial proton conductance in cells and mitochondria from the brown fat of cold-adapted and warm-adapted guinea pigs.

Cells and mitochondria were prepared from the brown adipose tissue of adult guinea-pigs adapted to either 4-7 degrees C or 22-25 degrees C. The cold-adapted cells displayed noradrenaline-stimulated, propranolol-sensitive respiration, but noradrenaline failed to increase the respiration of the warm-adapted cells. Purine-nucleotide-sensitive proton conductance was greater in cold-adapted mitochondria than in warm-adapted controls. At the same time cold-adapted mitochondria were extremely sensitive to the uncoupling effect of endogenous and infused fatty acids, and resembled the mitochondria from the brown adipose tissue of cold-adapted hamsters. Warm-adapted mitochondria were ninefold less sensitive, and resembled liver mitochondria. With cold-adapted, but not warm-adapted mitochondria, respiration increased proportionately to the rate of fatty acid infusion. It is concluded that the presence of the 32000-Mr proton conductance pathway is necessary for the expression of a high sensitivity to fatty acid uncoupling, suggesting that the fatty acids interact directly with this protein to modulate the proton conductance during the acute regulation of thermogenesis.

Acclimatization↗
PubMed1982

Fractionation of rat liver mitochondrial components after short treatments with Triton X-100.

1. When rat liver mitochondria are treated with the non-ionic detergent Triton X-100, the solubilizing effects of the latter are already maximal after a few seconds. 2. We have developed a filtration technique that allows a fast separation of the solubilized and non-solubilized fractions. By means of this technique, we have been able to show differences in the solubilizing effect of Triton X-100 according to the physiological state of mitochondria. 3. In addition, the filtrate may be subjected to further fractionation by ultracentrifugation, by which two filtrate subfractions, supernatant and pellet, may be obtained. 4. Filtrates, supernatants and pellets differ from each other, and from intact mitochondria, in phospholipid and polypeptide composition, and possess a characteristic distribution of enzyme activities; they are also different from the ultrastructural point of view. 5. It is concluded that the combined techniques of short detergent treatments and fast filtration are useful in structural and functional studies of mitochondria and in the selective solubilization of mitochondrial components.

Animals↗
PubMed1981

Constancy of liver lipid composition in two genera of toads after a short-term temperature acclimation.

The lipid and fatty acid composition of Bufo calamytes and Alytes obstetricans livers has been studied. Data for both species are similar, and resemble closely those published for Rana sculenta. Total lipids constitute 20-25% of the total liver dry weight; about one fifth of these are phospholipids; cholesterol makes up 1.7-2.2% of the total liver dry weight. The most abundant phospholipids are phosphatidylcholine and phosphatidylethanolamine. The various lipid cases differ in their fatty acid composition: neutral lipids contain high proportions of palmitoleic and linoleic acids; phosphatidylcholine is characterized by its contents in palmitic acid, whereas cardiolipin contains polyunsaturated fatty acids. Thermal acclimation of toads for 96 h produces but few changes in liver lipid composition.

Acclimatization↗