JoséM. Macarulla

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 .

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.

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.

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.

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

Abstract. 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 cortico- sterone 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.

Purification and characterization of the blue carotenoprotein from the caparace of the crayfish Procambarus clarkii (Girard)

Abstract The isolation, purification and characterization of a blue carotenoprotein isolated from the carapace of the crayfish Procambarus clarkii (Girard) are reported. The molecular weight of the complex has been determined by polyacrylamide gradient gel electrophoresis and gel filtration. Under unfavourable conditions the natural blue complex, designated the α-form (approx. M r 246 000), dissociated to a purple dimer, the β-form (approx. 41 600). Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated the β-form to contain two polypeptides, of molecular weight 19 200 and 21 400. The amino-acid composition of the natural protein is described and compared with those of similar carotenoproteins from other crustaceans. The complex contains six carotenoid molecules per molecule of protein (α-form) and the carotenoid has been identified by thin-layer chromatography, light-absorption spectroscopy, HPLC and mass spectrometry as all- trans -astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione), the three optical isomeric forms, (3 R ,3′ R )-, (3 R ,3′ S ) and (3 S ,3′ S ), being present in the ratio 20:21:58. The binding of the carotenoid, astaxanthin (absorption maximum in acetone at 470 nm) to the apoprotein results in a marked red spectral shift of about 165 nm, giving rise to absorption maxima at 635 nm and 585 nm for the α- and β-forms, respectively, in 50 mM phosphate buffer (pH 7.5). The λ max of any sample is dependent upon the relative ratio of α and β forms present.

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.

Structural characteristics of the carotenoids binding to the blue carotenoprotein fromProcambarus clarkii

A blue carotenoprotein from the crayfishProcambarus clarkii was extracted and purified. This carotenoprotein contains the carotenoid astaxanthin as a prosthetic group. In the present work we have identified by reconstitution, after removing the native carotenoid, some characteristics of the carotenoids that could bind to the apoprotein. The carotenoid must have two oxo groups at positions 4, 4′ and two hydroxyl groups at positions 3, 3′ the hexagonal or pentagonal end structure being indifferent. It has been proved that changes in the polyene chain structure such as triple bonds destroy this binding capacity.

A red carotenoprotein from the carapace of the crayfish, Procambarus clarkii

Abstract 1. 1. A red carotenoprotein ( λ max = 482 nm) containing astaxanthin and astaxanthin esters was extracted and purified from the carapace of the crayfish Procambarus clarkii . 2. 2. The extraction was achieved in the presence of Triton X-100. 3. 3. The red carotenoprotein had a mol. wt of ca. 140,000 (gel filtration). Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated only a single polipeptide of 8600 daltons. 4. 4. The red carotenoprotein contained 0.166 mg of protein, 0.833 mg of lipids, 0.032 μg of N -acetylglucosamine and 0.178 μg of astaxanthin per one mg of carotenoprotein complex.

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.