Biomedical subjects

J M Macarulla

Publications and source records attributed to J M Macarulla.

At least 37 records · Page 2Linked to original sources

PubMed1988

Lipid-protein interactions. The mitochondrial complex III-phosphatidylcholine-water system.

Bovine heart mitochondrial complex III (ubiquinol-cytochrome-c reductase) has been reconstituted into phosphatidylcholine bilayers and the effect of varying lipid/protein ratios on the structure and function of the protein has been examined. Electron microscopy, differential scanning calorimetry and Arrhenius plots of enzyme activity provide evidence that the protein is incorporated in an active conformation into pure phosphatidylcholine bilayers. At low lipid/protein ratios (e.g. 80:1 molar ratio) the protein exists in the form of aggregates. As the lipid proportion is increased, electron microscopy reveals the gradual formation of lipid bilayers; structures with the appearance of closed vesicles are seen at or above 300:1 phospholipid/protein molar ratios. Changes in enzyme activity as a function of lipid contents reveal a progressive increase in activity as more lipid is added, with a tendency to reach a saturation point. From the experimental data, a kinetic model is proposed, according to which the protein has an indefinite number of unspecific, independent and identical binding sites for phospholipids, the latter acting as essential enzyme activators. Varying lipid/protein ratios induce structural changes in complex III; visible spectra indicate changes in the polarity of the heme group environment, while Fourier-transform infrared spectroscopy suggests a change in the secondary structure of the protein as the lipid proportion is increased.

Animals↗

PubMed1988

Increased phospholipid methylation and glycogen phosphorylase activation after a single dose of dexamethasone in mouse liver.

Two different effects on phospholipid methylation and glycogen phosphorylase a activation were accomplished by treating mice livers with dexamethasone. A biphasic effect on the glycogen phosphorylase a was determined in the homogenated liver of dexamethasone-treated animals: The enzymatic activity increased between 45 to 90 min and decreased about 3 h. Moreover, the increased activity of glycogen phosphorylase a was also found in adrenalectomized mice. Also, the incorporation of ethanolamine into PE1) and PC1) after 15 min of hormone-administration was 77% and 270%, respectively, over the control values. The increase in phospholipid methylation was maintained during the first hour. After 4 h the hormone-treated animals also showed significant differences compared to the controls. Finally, studies of total lipid phosphorus content in the liver fail to show any differences between control and hormone-treated mice.

Adrenalectomy↗

PubMed1987

Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers.

The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues.

Animals↗

PubMed1987

An early effect of estradiol at hepatic level, previous to its protein synthesis activation.

Liver glycogen phosphorylase activity is increased before protein synthesis activation by estradiol. This effect is not blocked by antibiotics (actinomycin D and cycloheximide) inhibitor of protein synthesis. At times very similar to those of phosphorylase activation, cAMP levels are not enhanced, as would be expected, but slightly depleted. At similar times, cGMP levels are dramatically increased.

Animals↗

PubMed1987

Characterization of cortisol binding sites in chicken liver plasma membrane.

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.

Animals↗

PubMed1986

Phosphatidylethanolamine methyltransferase activity in chick liver microsomes.

The synthesis of phosphatidylcholine from phosphatidylethanolamine is carried out by chick liver microsomes (Gallus domesticus). Different concentrations of PE, NPE and NNPE were used as exogenous substrates. Saturation of the S-adenosylmethionine has been found for the three different reactions with or without exogenous substrate. Kinetic parameters have been determined for this enzyme system in chick liver microsomes. The three methyl reactions had a similar pH profile with an optimum at pH = 8. Divalent ions such as Ca2+ or Mg2+ did not stimulate the enzyme activity. The results suggest that the synthesis of phosphatidylcholine from phosphatidylethanolamine by chick liver microsomes exhibits a kinetic pattern with different aspects than that described for other animal or human preparations.

Animals↗

PubMed1986

Glycogenolytic effect of cortisol previous to its interaction with the cell nucleus in mouse liver.

Activities of hepatic glycogen synthase a and glycogen phosphorylase a have been studied in mouse liver at different times after an acute intraperitoneal administration of hydrocortisone. It has been observed an increase of glycogen synthase a activity and a decrease of glycogen phosphorylase a between 2 and 3.5 hours after cortisol injection. An early effect, previous to the synthase activation has been discovered. Cortisol caused an increase of glycogen phosphorylase a activity in mice 45 min after injection. This early effect of cortisol is independent of protein synthesis and it does not imply an increase in cAMP levels.

Animals↗

PubMed1986

Two independent effects of cortisol on chicken liver.

Activities of two key enzymes of glycogen metabolism have been measured after an acute administration of cortisol to 3d-old chickens. Glycogen synthase activity is enhanced 2-3 hours after a cortisol injection, and this activation is blocked by use of protein synthesis inhibitors. Glycogen phosphorylase activity is enhanced at an early stage, and this effect is not suppressed by protein synthesis inhibitors. Liver cAMP levels are not increased concomitantly with this early activation of glycogen phosphorylase; indeed they are depleted. These results point to the existence of an effect of cortisol previous to and independent of its nuclear interaction, and not mediated by an activation of the membrane adenylate cyclase.

Animals↗

PubMed1986

[Proteolytic susceptibility of alpha-amylase of the pancreas of swine deprived of its structural calcium].

Hog pancreas alpha-amylase (alpha-1-4-glucan-glucan hydrolase, E.C. 3.2.1.1) lost its structural calcium by action of EDTA at 20 degrees C. Enzymatic activity experimented a decrease whereas a big increase in proteolytic susceptibility to bovine pancreas trypsin (E.C. 3.4.4.4) was shown. Native alpha-amylase had an activity of 2,730 mg maltose/min X mg enzyme and a Km of 0.222% amylose, the activity of calcium depleted amylase being of 1,640 mg maltose/min X mg enzyme and Km 0.571% amylose. Simple methods for evaluating proteolytic susceptibility of alpha-amylase micro-amounts against trypsin action, and for the measurement of alpha-amylase activity in polyacrylamide rod gels were also described.

Animals↗

PubMed1985

Phospholipid oxidation catalyzed by cytochrome c in liposomes.

Oxidation of liposome phospholipids has been studied in the presence of cytochrome c. Sonicated vesicles of soya bean or egg-yolk lipids, or purified phospholipid preparations, were treated with oxidized cytochrome c at a 10:4 lipid/protein ratio (w/w). Lipid peroxidation was examined by oxygen polarography, gas-liquid chromatography (GLC) and the thiobarbituric acid test. Oxidized, but not reduced, cytochrome effectively catalyzes lipid oxidation under these conditions. Oxygen consumption and disappearance of unsaturated fatty acids follow closely similar patterns, the O2 consumption rate showing a maximum (1.53 mol O2/min per mol heme) shortly before fatty acid loss reaches its peak. GLC and O2 consumption data suggest that monohydroperoxides are the most abundant oxidized species in the system. The thiobarbituric acid reaction, however, appears only to be of qualitative value in peroxidation studies. In order to test the mechanism through which oxidation occurs in our system, the effect of liposome composition and the presence of antioxidants was tested, both on cytochrome c binding to bilayers and on O2 consumption. Oxidized and reduced cytochrome c bind the lipid bilayers with similar affinity, but only the oxidized form is active in autoxidation. Antioxidants do not modify either cytochrome c binding to sonicated liposomes. Lipid composition does influence considerably cytochrome binding, and O2 consumption is correspondingly altered. Studies with various antioxidants and inhibitors suggest that both free radicals and singlet oxygen may be involved in the process under study.

Animals↗

PubMed1985

Protein conformational transitions in sarcoplasmic reticulum membranes.

Fourier-transform infrared spectroscopic studies of sarcoplasmic reticulum proteins, in H2O and D2O, suggest that 10 mM ATP induces a conformational change in those proteins, increasing their contents in alpha-helical and beta-antiparallel structures. Ca2+ on the contrary, is seen to reduce the proportion of alpha-helix and increase the contribution of random coil.

Adenosine Triphosphate↗

PubMed1985

Laser light-scattering characterization of mitochondrial complex III-Triton X-100-phospholipid mixed micelles.

Bovine-heart mitochondrial complex III was purified in the presence of Triton X-100, and the size and shape of the resulting protein-surfactant-phospholipid mixed micelles were investigated by laser light-scattering. The protein appears to be present in the form of a dimer, irrespective of temperature (between 25 and 40 degrees C) and protein concentration (between 0.5 and 5 mg/ml). The molecular weight of the micelle increases with temperature from 600 000 (25 degrees C) to 692 000 (40 degrees C). The variation of the solvent second virial coefficient in this temperature range suggests that, with increasing temperature, some of the free surfactant molecules become integrated in the mixed micelles. The average quadratic radius of gyration of these is of 42 +/- 5 nm, corresponding in our case to an ellipsoidal shape.

Animals↗

PubMed1984

Infrared spectroscopy of phosphatidylcholines in aqueous suspension. A study of the phosphate group vibrations.

The 1000-1300 cm-1 region of the infrared spectrum of dipalmitoylphosphatidylcholine (DPPC) and other phosphate-containing molecules has been studied by the Fourier-transform technique. Three absorption bands have been assigned to various vibrational modes of the DPPC phosphate group, with maximum wavenumbers at 1060, 1086 and 1222 cm-1. These values are the same above and below Tc of the phospholipid. Dehydration produces band-shifts toward higher wavenumbers .

Fourier Analysis↗

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↗

PubMed1984

Cholesterol biosynthesis in chicken liver: effect of triiodothyronine.

The effect of a single injection of L-triiodothyronine (T3) on the levels of liver cholesterol and liver 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) has been studied in chicken. A minimal dose of 8 micrograms/animal produces its maximal effects after 12 h. T3 enhances both HMGR activity and cholesterol levels in liver. Both effects are suppressed when protein synthesis inhibitors (actinomycin D or cycloheximide) are administered along with the hormone. Solubilized and membrane-bound HMGR activities are equally influenced by the hormone. The in vitro addition of T3 does not affect the HMGR activity. It is concluded that T3 increases cholesterol biosynthesis in chicken liver through the activation of de novo protein synthesis.

Animals↗

PubMed1984

Effect of a single dose of T3 on the incorporation of (Methyl-14C) methionine into phosphatidylcholine and lysophosphatidylcholine in chick liver.

The preliminary effect of a single dose of T3 (Triidothyronine) on the incorporation of (Methyl-14C) Methionine into liver phosphatidylcholine, lysophosphatidylcholine and methylated precursors was studied in chick liver from 0.25 to 6 hours. Chicks received a T3 dose intraperitoneally 5 h prior labeled compound administration. It can be deduced that the hormone effect is similar in phosphatidylcholine and lysophosphatidylcholine. In our conditions, the preliminary hormone effect is maximum 5 1/2 h after T3 injection. The levels of methylated precursors have been studied by radioactivity and lipid P determinations; these levels are constant throughout the time course experiment. We conclude that the increase in methyl group incorporation is due to a hormone-dependent increase in phospholipid turnover.

Animals↗

PubMed1984

An interaction of testosterone with cell membranes.

The existence of an early effect of testosterone, prior to the effects dependent upon interaction between the hormone-citosolic receptor complex and the cellular nucleus, has been explored in the present paper, in 3-day old chickens. Liver glycogen phosphorylase activity is increased before protein synthesis activation, and furthermore this effect is not blocked by antibiotics (actinomicin D and cycloheximide) inhibitor of protein synthesis. When liver phosphorylase is activated, cAMP levels are not enhanced, as would be expected, but deeply depleted. The hypothesis of a phosphorylase-kinase activation due to an increase in the intracellular Ca++ concentration is considered.

Animals↗