Purification of a cortisol binding protein from hepatic plasma membrane.

A cortisol binding protein from rat liver plasma membranes has been solubilized in active form by using the zwitterionic detergent CHAPS. Two types of binding sites have been characterised in both native and solubilized membranes. The first is of high affinity and low binding capacity (12 nM; 946 fmol/mg) and the other one is of low affinity and high capacity of binding (344 nM; 12677 fmol/mg) for solubilized membranes. The purified material retained a binding activity comparable to that displayed by the original membrane. The specific binding activity was enriched about 12700-fold, with an 8% yield. Analysis of the purified preparation on sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed two protein subunits with molecular mass of 52000 and 57000 Da. The new cortisol-specific binding membrane protein could be related to the nongenomic effects previously described for this hormone.

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.

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 .

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.

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.

Membrane-surfactant interactions. The effect of Triton X-100 on sarcoplasmic reticulum vesicles.

The effect of Triton X-100 on purified sarcoplasmic reticulum vesicles has been studied by means of chemical, ultrastructural and enzymic techniques. At low detergent/membrane ratios (about 1 Triton X-100 per 60 phospholipid molecules) the only effect observed is an increase in vesicle permeability. Higher surfactant concentrations, up to a 1:1 detergent/phospholipid ratio, produce a large enhancement of ATPase activity. Membrane solubilization occurs as a critical phenomenon when the surfactant/phospholipid molar ratio reaches a value around 1.5:1, corresponding to 2 mumol Triton X-100/mg protein. At this point, the suspension turbidity drops, virtually all the protein and phospholipid is solubilized and every organized structure disappears. Simultaneously, a dramatic increase in the specific activity of the solubilized ATPase is observed. The sudden solubilization of almost all the bilayer components at a given detergent concentration is attributed to the relative simplicity of this membrane system. Solubilization takes place at the same surfactant/membrane ratio, at least between 0.5 and 4 mg membrane protein/ml. The non-solubilized residue seems to consist mainly of delipidized aggregated forms of ATPase.

Dual effects of ATP on phosphatidylinositol breakdown in rat hepatocyte membranes.

The mechanisms whereby adenosine-5'-triphosphate (ATP) regulates the inositol phospholipid-signalling system were studied in rat hepatocytes. Intact hepatocytes respond to extracellular ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP and weakly to guanosine-5'-triphosphate (GTP), but not to other purine nucleotides (GDP or AMP). This is consistent with the idea that a P2 purinergic receptor is coupled to the phosphatidylinositol metabolism in these cells. Partially purified plasma membranes prepared from myo-[3H]inositol prelabelled hepatocytes exhibit a phosphatidylinositol-4,5-bisphosphate phospholipase C activity sensitive to ATP, ATP gamma S and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S). Moreover the GTP gamma S effect is greatly enhanced by ATP and ATP gamma S. These potentiating effects differ according to the adenylnucleotide considered. ATP produces (1) an increase in the GTP gamma S-PLC sensitivity, (2) a potentiation of the phospholipase C (PLC) response induced by maximal dose of GTP gamma S, and (3) an increase in the inositol lipids pools. At variance, ATP gamma S, a nonhydrolysable analogue of ATP, only increases the PLC-sensitivity towards GTP gamma S. These results may signify that ATP stimulates inositol phosphate accumulation via at least two distinct mechanisms (i) a direct activation of a P2 purinergic receptor coupled to a PLC via a GTP binding protein and (ii) a stimulation of the phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinases which increased the pool of phospholipase C substrates.

A simple unified explanation of the red-ox and acid-base concepts.

Some classical examples of the habitual physico-chemical processes are studied and a unifying conclusion is reached. In the red-ox processes, the atoms or groups of atoms which gain electrons according to their respective oxidation numbers are oxidizing, i.c. if the bindings are considered pure ionic, and in the acid-base processes the atoms or groups of atoms which only gain electrons according to their formal charge are acids, i.e. if the bindings are considered pure covalent.

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.

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.

[The effect of the thyroid hormone triiodothyronine on the incorporation of 32P into brain phospholipids in rat and chicken (author's transl)]. / Efecto de la triiodotironina sobre la incorporación de P32 a fosfolípidos cerebrales de pollo y rata.

The incorporation of 32P to brain phospholipids has been studied, in chicken as well as in rat. Both animal species were at different stage of brain development, although degrees of psychomotive development were identical. It is observed that the effect of triiodothyronine is more pronounced when the degree of brain myelination is smaller. The radioactive precursor is frequently incorporated to choline and ethanolamine phospholipids, and this incorporation is higher in hyperthyroid animals with a low degree of myelination. The triiodothyronine activates, in both animal species, the salvage pathway of phospholipid biosynthesis. This activation takes place 36 h before hormone action in chicken, while in rat, the most significant differences occur 36 h after hormone action.

Characterization of specific corticosterone binding sites in adrenal cortex plasma membrane and their localization by autoradiographic studies.

Specific corticosterone binding to calf adrenal cortex plasma membrane was measured using the biologically active radioligand [3H]corticosterone. Corticosterone binding was found to be time-dependent, saturable and reversible, and was reduced by more than 70% when membranes were pretreated with proteases. The population of corticosterone binding sites in calf adrenal cortex plasma membrane was homogeneous and displayed the following characteristics: equilibrium dissociation constant Kd = 77 +/- 8 nM and maximum specific binding capacity Bmax = 70,378 +/- 6,385 fmol/mg protein. The relative affinities of several structural analogues of steroids were deduced from competition assays. From these experiments we can conclude that the plasma membrane binding site characterized is selective for corticosterone and progesterone derivatives, and different from nuclear glucocorticoid, mineralocorticoid, estrogen and progestin receptors. Likewise, this corticosterone binding site is independent of mineralocorticoid and Na+, K(+)-ATPase digitalis receptors. From autoradiographic studies we suggest these corticosterone binding sites are located in the whole adrenal cortex.

[Effect of triiodotyronine on liver glycogen in gallus domesticus (author's transl)]. / Efecto de la triyodotironina sobre el glucógeno hepático en Gallus domesticus.

The effect of single injections of triiodotyronine (T3) on liver glycogen levels of Gallus domesticus has been studied at the embryonary and postnatal stages. In starved newborn chickens, after the administration of 4 microgram of T3, an important decrease in liver glucogen is observed between 4 and 10 hours after the injection. In a series of experiments following the chicken development, a gradual increase in glycogen can be seen, until the sixth day after eclosion, with the only exception of newborn chickens. T3 has a gradual and significant effect from the 17 th. incubation day onwards producing a decrease in liver glycogen to values lightly inferior to 50% of the control.

[Effects of T3, T4 and methyltiouracile short-term treatments on plasma and liver lipid levels in rats (author's transl)]. / Efectos de la administración a corto plazo de T3, T4 y metiltiouracilo sobre los niveles de lípidos en hígado y plasma de rata.

The effects of short-term administration of thyroid hormones (T3 and T4) and methyltiouracile (MTU) on liver and plasma lipid levels in female rats have been comparatively studied T3 increases the levels of all the studied liver lipids and decreases the plasma lipids. T4 produces smaller and even, in some cases (liver triglycerides and cholesterol esters), opposite effects. The administration of MTU produces, in general, opposite effects to T3 and T4.

[Effect of the thyroid hormone, triiodothyronine on fatty acid chain elongation of mitochondrial phospholipids (author's transl)]. / Efecto de la hormona tiroidea triiodotironina sobre la elongación de los ácidos grasos de fosfolípidos mitocondriales.

The effect of the Thyroid Hormone Triiodothyronine (T3) on composition of Fatty Acid of Mitochondrial Phospholipids is studied. In animals (Gallus domesticus var. Cornis and White Roc.) treated with an injection of T3 containing 0.04 microgram/g body weight, increase length and insaturation increases of the hydrocarbon chains of fatty acids in mitochondrial phospholipids are observed in relation to the corresponding controls. This elongation is maximum at 14 hours after treatment with the hormone. Animals treated with T3 showed a higher proportion of stearic and eicosatrienoic acids than controls. This increase is accompanied by an equivalent decrease of the palmitic and oleic acids, their respective biosynthetic precursors. The highest proportion of long chain and unsatured fatty acids can be observed in phosphatidylethanolamines and phosphatidylcholines.

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles.

The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca++ accumulation in SR vesicles at detergent concentrations below 10(-3) M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca++ accumulation in the concentration range between 10(-4) M and 2.5 X 10(-2) M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca++-ATPase activity up to a 10(-3) M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.

An early effect of cortisol, previous to its glycogenogenic action.

Cortisol produces a glycogenogenic effect 5 hours after intraperitoneal injection to 3 day old chicks. This effect is dependent on protein synthesis because it can be blocked by antibiotics such as actinomycin D. On the other hand, there is a previous glycogenolytic effect 45 minutes after cortisol administration which is independent of protein synthesis. Thyroid hormones produce a similar early effect as has been previously shown. However, the observed glycogenolysis after cortisol injection is not correlated with an enhancement in the liver cAMP levels.

Effect of acute administration of triiodothyronine in chicken. Liver glycogen depletion and amino acid incorporation to proteins.

Single injections of thyroid hormone (T3) produce liver glycogen depletion in chickens. This effect cannot be suppressed by protein synthesis inhibitors and is previous to the hormone-induced increase in protein synthesis.

Carotenoids from marigold (Tagetes erecta) petals and their esterified fatty acids.

The main carotenoid in Tagetes erecta petals is lutein, which is found either free or esterified to one or two fatty acids. Column and thin-layer chromatographic methods are described for the separation of the different lutein species, and compared to other, previously published, techniques. In addition, fatty acid distributions are given for whole marigold plants, crude carotenoid extracts and purified mono and diesterified luteins obtained from fresh petals as well as from commercial powdered petal preparations.

[Proteolytic susceptibility of alpha-amylase of the pancreas of swine deprived of its structural calcium]. / Susceptibilidad proteolítica de la alpha-amilasa de páncreas de cerdo privada de su calcio estructural.

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.