Investigation of phycocyanin-membrane interaction: Promotion of membrane interactions

• 1.1. In a continuing investigation of phycocyanin-membrane surface interaction, fluorescence quenching experiments were performed with a mixture of two populations of fluorescence probe-encapsulated phospholipid bilayer vesicles in the presence and absence of phycocyanin.
• 2.2. These membrane vesicles were prepared with 1,2-dimyristoyl phosphatidylcholine (DMPC), cholesterol and a probe molecule.
• 3.3. A fluorophore was encapsulated in one population of membrane vesicles, while a quencher was encapsulated in another population of membrane vesicles.
• 4.4. The result was compared with those of experiments in the presence of other biomolecules, including albumin, cytochrome c, hemoglobin, myoglobin or RNA.
• 5.5. Interestingly, a one-third reduction of the fluorescence intensity was observed in the mixture of these two populations of membrane vesicles in phycocyanin's presence.
• 6.6. In contrast, the other biomolecules caused no significant reduction in the fluorescence intensity.
• 7.7. These findings were evidence of a phycocyanin-induced membrane perturbation.
• 8.8. This was further demonstrated by a phycocyanin-induced change in the thermotropic behavior of DMPC vesicles, as measured by differential scanning microcalorimetry.
• 9.9. Such a unique property of phycocyanin is believed to be associated with its known membrane surface-interacting character.
• 10.10. A possible phycocyanin-modulated membrane-membrane interaction was discussed.

     

Cell surface labeling by iodine monochloride

• 1.1. Human red cell membranes were trace iodinated with [¹²⁵I]ICI and the distribution of label in membrane components examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 2.2. With intact erythrocytes over 84% of the bound iodine was associated with the membrane.
• 3.3. Two membrane components accounted for almost all of the label, band 3 and PAS-1.
• 4.4. Spectrin was not labeled in resealed ghosts.

     

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

• 1.1. Four GTP-binding proteins (23–27 kDa) were identified in membranes from PC12 cells by [α³²P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels.
• 2.2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K⁺-depolarization or even after addition of C²⁺to digitonin-permeabilized cells.
• 3.3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca²⁺.
• 4.4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity.
• 5.5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane.
• 6.6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

     

Effect of LCAT on HDL-mediated cholesterol efflux from loaded EA.hy 926 cells

• 1.1. Human endothelial cells (EA.hy 926 line) were loaded with cholesterol, using cationized LDL, and the effect of lecithin:cholesterol acyltransferase (LCAT) on cellular cholesterol efflux mediated by high density lipoproteins (HDL) was measured subsequently.
• 2.2. In plasma, lecithin:cholesterol acyltransferase (LCAT) converts unesterified HDL cholesterol into cholesteryl esters, thereby maintaining the low UC/PL ratio of HDL. It was tested if further decrease in UC/PL ratio of HDL by LCAT influences cellular cholesterol efflux in vitro.
• 3.3. Efflux was measured as the decrease of cellular cholesterol after 24 hr of incubation with various concentrations of HDL in the presence and absence of LCAT. LCAT from human plasma (about 3000-fold purified) was added to the cell culture, resulting in activity levels in the culture media of 60–70% of human serum.
• 4.4. Although LCAT had a profound effect on HDL structure (UC/TC and UC/PL ratio's decreased), the enzyme did not enhance efflux of cellular cholesterol, using a wide range of HDL concentrations (0.05–2.00 mg HDL protein/ml).
• 5.5. The data indicate that the extremely low unesterified cholesterol content of HDL, induced by LCAT, does not enhance efflux of cholesterol from loaded EA.hy 926 cells. It is concluded that the HDL composition (as isolated from plasma by ultracentrifugation) is optimal for uptake of cellular cholesterol.

     

Lysis of resealed erythrocyte ghosts by photoactivated tetrapyrroles: Estimation of photolesion dimensions

• 1.1. Resealed erythrocyte ghosts containing Na⁺ and glucose-6-P (G6P) as markers of membrane integrity were used as a model system for probing the damaging effects of photoactivated tetrapyrroles on cell membranes.
• 2.2. Continuous blue-light irradiation of bilirubin (BR)-sensitized and protoporphyrin (PP)-sensitized ghosts made them progressively more permeable to Na⁺ the cation emerging well ahead of G6P.
• 3.3. G6P efflux occurred abruptly after a lag period and resembled an all-or-none process.
• 4.4. These and other results suggest that a relatively subtle structural modification (possibly in some crucial protein(s) is sufficient for Na⁺ release, whereas gross disruption of the bilayer (probably by free-radical lipid peroxidation) is necessary for G6P release.
• 5.5. The dimensions of the G6P-releasing photolesions were estimated by density floatation centrifugation, using saccharides of increasing molecular size. Both BR and PP produced pores > 11 Å but <42 Å in diam, which is considerably smaller than the size range estimated in hypotonically lysed ghosts.

     

Na+-independent sugar uptake by rat intestinal and renal brush border and basolateral membrane vesicles

• 1.1. Uptake of [¹⁴C]-labelled d-glucose, l-arabinose and d-fructose by intestinal and renal brush border and basolateral membrane vesicles was studied in the absence of Na⁺ .
• 2.2. The Na⁺-independent d-glucose transport system in these membrane vesicles was saturable, sensitive to phloretin, stereospecific and accessible only to d-glucose and d-galactose.
• 3.3. Na⁺-independent l-arabinose transport was not saturable even when its concentration was raised to 300 mM and it was insensitive to phloretin.
• 4.4. Na⁺-independent d-fructose transport demonstrated saturation kinetics with only renal brush border membrane vesicles, but it was not inhibited by either phloretin or phlorizin.
• 5.5. These studies indicated that the Na⁺-independent carrier-mediated d-glucose/d-galactose transport system of intestinal and renal brush border and basolateral membranes is clearly not shared by other monosaccharides.

     

The isolation of skeletal muscle plasma membranes from rainbow trout (Salmo gairdneri)

• 1.1. Plasma membranes were isolated from caudal flank skeletal musculature of rainbow trout by discontinuous sucrose gradient centrifugation.
• 2.2. Na⁺−K⁺-ATPase was enriched 8-fold and 5′-nucleotidase activities 4-fold in a fraction isolated at the 8–25% sucrose interface.
• 3.3. A cholesterol: phospholipid ratio of 0.37 in the plasma membrane fraction was 85% greater than that observed in adjacent subcellular fractions.
• 4.4. Electron microscopy provided morphological confirmation of enrichment and integrity of skeletal muscle plasma membranes at the 8–25% sucrose interface.

     

Functional changes of rat brain mitochondrial enzymes induced by monomeric cholesterol

• 1.1. The binding of [¹⁴C]cholesterol into rat brain mitochondrial membranes follows an exponential path described by the general formula y = a.e^bx. [¹⁴C]cholesterol glucoside binding has a sigmoidal character where the “best-fit” curve of this type of binding is the one described by the Hill equation with Hill coefficient h = 2.06. These findings suggest a positive cooperativity in the binding of both compounds into rat brain mitochondrial membranes.
• 2.2. The specific activity of the outer mitochondrial membrane enzyme monoamine oxidase was linearly decreased at different concentration of cholesterol or its glucoside.
• 3.3. The specific activity of the inner mitochondrial membrane enzyme succinate-cytochrome c reductase was linearly decreased, while that of Rotenone-sensitive NADH-cytochrome c reductase was exponentially increased, at different concentrations of cholesterol.
• 4.4. These results are discussed in terms of specific interactions of cholesterol with constituent mitochondrial membrane lipids and their implications for deviations from normal neuronal function.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

     

The influence of calcium and magnesium on sea bass (Dicentrarchus labrax) intestinal brush border membrane purification and activity

• 1.1. The activity of brush border enzymes (alkaline phosphatase, maltase, sucrase, trehalase, leucine amino peptidase) was higher in purified membranes prepared with calcium. The contamination of these membranes with basolateral membranes was also lower (1.27 for Na-K-ATPase activity ratio).
• 2.2. The extraction of brush border lipids was carried out according to Folch adapted method. Two dimensional thin layer chromatography was used to separate the phospholipidic fractions. Fatty acids of phospholipids were analysed using gas chromatography after acid transmethylation (column SP 2330).
• 3.3. Phospholipids are composed of phosphatidylcholine (PC: 33%), phosphatidylethanolamine (PE: 30%), sphingomyeline (SM: 21%), phosphatidylserine (PS: 14%) and phosphatidylinositol (PI: 2%). 4. PC, PE and PS are characterized by high levels of unsaturated fatty acids (monounsaturated MUFA: 21.5% and polyunsaturated PUFA: 34.9%). The most abundant PUFA belong to the (n-3) family [18:3 (n-3), 20:5 (n-3) and 22:6 (n-3)].
• 4.5. Fatty acids from sphingomyelin of purified membranes have low proportions of PUFA (13.5%) but higher proportions of MUFA (39.5%).
• 5.6. No specific differences were found between calcium and magnesium prepared membranes.
• 6.7. The low content in LPC and the absence of LPE confirmed the absence of major structural lipids transformation during the membrane purification with calcium or magnesium.
• 7.8. Glycine transport was measured during 10 sec at different temperatures using the rapid filtration technique. Glycine transport was higher with Na⁺ than with K⁺. In the presence of Na⁺, this transport increases with temperature.
• 8.9. Arrhenius curves were mono phasic without obvious breakpoint and indicated no phase transition in the lipid bilayer.
• 9.10. A significant Na⁺ dependent glycine transport has been characterized at low temperatures (0°C) which suggests a possible role of membrane polyunsaturated fatty acids in the control of glycine transport.

     

In vitro modification of cholesterol/ phospholipid ratio of enterocytes brush border membrane and its effect on l-leucine accumulation

• 1.1. Incubation of intestinal segments from chicken jejunum in liposome suspensions with different cholesterol/phospholipid ratios lead to change in the enterocytes brush border membrane cholesterol/phospholipid molar ratio.
• 2.2. The fatty acid composition, total amount and the ratios of the individual phospholipids of enterocytes brush border membrane were not affected.
• 3.3. The change in the cholesterol/phospholipid molar ratio had no effect on l-leucine accumulation in the enterocytes.

     

The effects of cholesterol feeding on the membranes of liver cells and on the cholesterol metabolism in the rat

• 1.1. Feeding of rats with a 2% cholesterol diet for 6 weeks increased the serum cholesterol concentration. The activity of lecithin cholesterol acyltransferase was also increased during the feeding time.
• 2.2. The activities of aspartate aminotransferase and alanine aminotransferase remained on a constant level during the experiment on rats having cholesterol in their diet. Omitting cholesterol from the diet enhanced the activities of both enzymes and the increase in alanine aminotransferase activity was more pronounced.
• 3.3. The activity of alkaline phosphatase was on higher level during the whole experiment in the rats having cholesterol in the diet than in those fed a cholesterol-free diet.
• 4.4. Present data suggest that excluding cholesterol from the diet labilizes the membranes of hepatocytes and facilitates the release of aspartate and alanine aminotransferases in the blood.

     

Properties and topology of enzymes methylating phosphatidylethanolamine to phosphatidylcholine in sarcoplasmic reticulum

• 1.1. The synthesis of phosphatidylcholine (PC) by stepwise methylation of phosphatidylethanolamine (PE) is carried out by two enzymes in sarcoplasmic reticulum (SR) membrane of rabbit fast-twitch skeletal muscles.
• 2.2. Two methyltransferases (Met I and Met II) have a different pH optimum and affinity for methyl donor—5-adenosyl-L-methionine (SAM).
• 3.3. Met I is an integral SR membrane protein which active site faces the cytoplasmic surface of the membrane.
• 4.4. Met II is a peripheral, loosely bound protein, localized mainly on the extracytoplasmic (luminal) part of the SR membrane.

     

Factors influencing lipid levels and lecithin:cholesterol acyltransfer in plasma of the lizard,Tropidurus torquatos

• 1.1. Lipid concentrations and lecithin:cholesterol acyltransferase (LCAT) activity in the plasma Tropidurus torquatos were remarkably variable.
• 2.2. Both lipid levels and LCAT activity were highest for lizards collected during the early rainy season (March–April) than during other seasons, and were higher for females than for males.
• 3.3. Plasma lipid levels and LCAT activity were significantly and inversely correlated with body weight (age) of male lizards, this being associated with an apparent change to an herbivorous diet in older males.
• 4.4. During prolonged fasting, plasma lipid levels and lecithin:cholesterol acyltransfer (LCAT) and hepatic phospholipids were markedly reduced.
• 5.5. LCAT activity in plasma of fasted and non-fated lizards was significantly correlated with the molar proportion of PC to UC, suggesting that the apparent low LCAT in plasma of fasted lizards is partly due to depletion of PC in the lipoprotein substrates.

     

Effect of caffeine,theophylline and nicotine on d-glucose and folate transport in rat jejunal brush border membrane vesicles

• 1.1. Nicotine at 10 mM, but not caffeine or theophylline, reduced by 20% the overshoot of the Na⁺-dependent d-glucose transport in ratjejunal brush border membrane vesicles.
• 2.2. Since nicotine did not affect the transport of Na⁺, its inhibition on Na⁺-dependent d-glucose transport must be due to a direct effect upon the d-glucose transport system.
• 3.3. Folate transport in these membrane vesicles was found to a be a free diffusion process at pH 7.4.
• 4.4. Neither caffeine, theophylline nor nicotine has any effect on folate transport.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

Spingophosphonolipids in microsomes of Diplodon delodontus. Distribution and effect on the membrane microviscosity

• 1.1. The distribution of ceramide aminoethylphosphonate (CAEP) in microsomal membranes obtained from different tissues of the bivalve mollusc Diplodon delodontus was determined.
• 2.2. The concentration of CAEP reached from 9 to 19% of the total microsomal polar lipids, depending on the kind of tissue.
• 3.3. Palmitic acid was the main fatty acid in the ceramide moiety, followed by stearic and eicosamonoenoic acids.
• 4.4. Artificial membranes were prepared with microsomal phospholipids or phospholipids plus sterols, with and without the addition of CAEP.
• 5.5. It was shown that the phosphonate confers minor mobility to the membranes. This effect is more effective when the membrane contains the natural sterols and the phospholipids are unsaturated.

     

Ultrastructure of maturing fish (Oreochromis niloticus) and snake (Waglerophis merremii) erythroid cells with regard to hemoglobin biosynthesis

• 1.1. Fish and snake immature erythrocytes were submitted to a comparative ultrastructural study, analysing changes in organelles involved in hemoglobin (Hb) biosynthesis.
• 2.2. Iron uptake occurs probably via transferrin, and ferruginous compounds accumulate as siderosomes, taken as iron sources for heme biosynthesis, later on caught by a double lamella.
• 3.3. Mitochondrial membrane of the inner camera differentiates to lamellated bodies that, sucessively, give rise to expansions for ferruginous material and globin chains captation, constituting prehemosomal vesicles, which become condensed vesicles, followed by prohemosomes.
• 4.4. Through an internal membrane rearrangement, prohemosomes change to hemosomes wherein, hypothetically, heme and the globin chains assembly may occur.
• 5.5. In both fish and snake erythroid cells, all stages for hemosomegenesis are similar to the stages found in erythroid cells of other vertebrate species, including humans, except that fish cells often present single organelles of still unknown function, void of internal membrane.
• 6.6. Through electrophoresis of the respective supernatants obtained after osmotical lysis of the organellar fractions, it was shown that fish hemosomes contain three Hb patterns, while snake hemosomes present two patterns.

     

Localization of the glucocorticoid receptor in rat liver cells: Evidence for plasma membrane bound receptor

• 1.1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction.
• 2.2. After in vivo administration of [³H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes.
• 3.3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol).
• 4.4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes.
• 5.5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.

     

Plasma membrane: Can its structure and function be modulated by dietary fat?

• 1.1. Compositional analysis of plasma membranes from rats fed nutritionally adequate diets different in fatty acid composition establishes that fundamentally different dietary fat intake results in alteration in structural lipid composition of plasma membranes in brain, liver and the intestinal mucosa.
• 2.2. Dietary differences in fatty acid intake altered the fatty acyl tail composition of plasma membrane phospholipids in brain, liver and intestinal mucosa.
• 3.3. Diet altered the phospholipid profile observed in brain synaptosomal and liver plasma membrane.
• 4.4. Feeding high vs low polyunsaturated to saturated fat diets for 7 days altered the fatty acid composition of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and mono-glucosylceramide isolated from plasma membrane of the intestinal mucosa