Interaction of T-2 toxin with murine lymphocytes

T-2 toxin is taken up by lymphocytes in 10–15 min in a saturable manner. Uptake is dependent on temperature and partially on the availability of energy. Approx. 10⁵ molecules of T-2 toxin are bound per cell, having a mean affinity constant, K_a = 1.6·10⁷ M^?1. The toxin is rapidly dissociated from the cell to leave approx. 10–15% of the original loading in 1 h. It is concluded that T-2 toxin uptake and release do not follow conventional mechanisms.     

The Interaction of Flavonoids with Membranes: Potential Determinant of Flavonoid Antioxidant Effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2′-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2′-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid–water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Effect of fatty acids on plasma membrane lipid dynamics and cation permeability in neuroblastoma cells

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3′-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from ?51 mV to ?36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using ⁸⁶Rb⁺ as a radioactive tracer for K⁺, demonstrated that the depolarization was not caused by changes in (Na⁺ + K⁺)-ATPase-mediated K⁺ influx or in the transmembrane K⁺ gradient. The permeability ratio ($\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ was shown by ²⁴Na⁺ and ⁸⁶Rb⁺ flux measurements to be due to both an increase of the Na⁺ permeability and a decrease of the K⁺ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.     

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [³⁵S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.     

The pyruvate: Ferredoxin oxidoreductase in heterocysts of the cyanobacteriuim Anabaena cylindrica

Heterocyst preparations have been obtained which actively perform nitrogen fixation (C₂H₂ reduction) and contain the enzymes of glycolysis and some of the tricarboxylic acid cycle. Pyruvate: ferredoxin oxidereductase has been unambiguously demonstrated in extracts from heterocysts by the formation of acetylcoenzyme A, CO₂ and reduced methyl viologen (ferredoxi) from pyruvate, coenzyme A and oxidized methyl viologen (ferredoxin) as well as by the synthesis of pyruvate from CO₂, acetylcoenzyme A and reduced methyl viologen. Pyruvate supports C₂H₂ reduction by isolated heterocysts, however, with lower activity than Na₂S₂O₄ and H₂. α-Ketoglutarate: ferredoxin oxidoreductase is absent in Anabaena cylindrica, confirming that the organism has an incomplete tricarboxylic acid cycle.     

Purification of a membrane-derived proteinase capable of activating a galactosyltransferase involved in volume regulation

UDPgalactose:sn-glycerol-3-phosphate α-D-galactosyltransferase (IFP-synthase, EC 2.4.1.96) shows low activity in extracts prepared from standard volume cells of Poterioochromonals malhamensis under certain conditions. This inactive enzyme has been partially purified by chromatography on DEAE-cellulose, Sephadex G-150 and α-lactalbumin-agarose. It can be activated by an auxiliary enzyme which can be eluted from membranes and which has been purified to homogeneity by chromatography on DEAE-Sephacel and immobilized hemoglobin and fetuin. The activating enzyme is inhibited by chymostatin, antipain and diisopropylfluorophosphate and does not require divalent ions. It consists of a single peptide chain of molecular weight 46 000, can split certain proteins and appears to be a serine proteinase operating around a pH of 6.0. The activating proteinase is irreversibly generated in the crude homogenates on addition of Ca²⁺ and also shows increased activity shortly after cell shrinkage. This might indicate that it represents one of the possibilities to render the galactosyltransferase active as a result of the physiological stimulus.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates.

1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.

2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM⁻¹ · cm⁻¹) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.

3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min⁻¹ · mg⁻¹ of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.

4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Proteins from crown gall tissue labelled in vivo with [³²P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total ³²P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.     

Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: a fluorescence study using Pep-1

Cell-penetrating peptides (CPPs) are able to translocate across biological membranes and deliver bioactive proteins. Cellular uptake and intracellular distribution of CPPs is commonly evaluated with fluorescent labels, which can alter peptide properties. The effect of carboxyfluorescein label in the Lys-rich domain of the amphipathic CPP pep-1, was evaluated and compared with non-labelled pep-1 in vitro and in vivo. A reduced membrane affinity and an endosomal-dependent translocation mechanism, at variance with non-labelled pep-1, were detected. Therefore, the charged domain is not a mere enabler of peptide adsorption but has a crucial role in the translocation pathway of non-labelled pep-1.     

Studies on the ferrochelatase activity of mitochondria and submitochondrial particles with special reference to the regulatory function of the mitochondrial inner membrane

The question addressed in the title was examined by measuring fluorescence emission spectra and light-induced fluorescence-yield changes of chloroplasts which had been frozen to ?196 °C rapidly, as very thin samples adsorbed into substrates which were plunged directly into liquid nitrogen, or slowly by the cooling action of liquid nitrogen through the wall of the cuvette. Contrary to previous reports, we found that the rate of cooling had no influence on the shape of the emission spectrum, the extent of the variable fluorescence or the fraction of the absorbed quanta which are delivered initially to Photosystem I.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

The interaction of flavonoids with membranes: potential determinant of flavonoid antioxidant effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2'-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2'-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid-water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

PhoE protein pores in the outer membrane of Escherichia coli K-12 not only have a preference for Pi and Pi-containing solutes but are general anion-preferring channels

Previous studies on the question of whether the PhoE protein pore has a preference for P_i and P_i-containing solutes only or whether it constitutes a general anion-preferring channel, have not given an unequivocal answer either because the presence of the phosphate binding protein was not ascertained or because only arsenate was tested as a non P_i-containing control solute. Permeability properties of PhoE, OmpF and OmpC protein pores for negatively charged solutes were measured in vivo in the presence of phosphate-binding protein. It appeared that the PhoE protein pore is the most efficient channel for the three tested solutes phosphate, succinate and sulphate. Conditions were established to measure the frequency of ethyl methane sulphonate induced mutations as a function of the presence of pore proteins. These results indicate that PhoE protein also forms the most efficient channel for ethyl methane sulphonate. We conclude that the preference of the PhoE protein pore is not restricted to P_i and P_i-containing solutes but also concerns several other negatively charged solutes.     

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [¹²⁵I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [¹²⁵I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Lipid-protein interactions of the human erythrocyte concanavalin a receptor in phospholipid bilayers

The interaction of the human erythrocyte concanavalin A receptor (a subpopulation of Band 3) with phospholipids has been investigated using differential scanning microcalorimetry of reconstituted vesicles prepared by detergent dialysis. The mean diameter of dialyzed phospholipid vesicles jumps dramatically on inclusion of the concanavalin A receptor and then increases linearly with the fraction of protein in the bilayer. The glycoprotein has a dramatic effect on the phospholipid gel to liquid-crystalline phase transition, and ΔH decreases linearly with increasing mole fraction of protein up to a protein/lipid mole ratio of around 1:1160. Extrapolation of this data indicates that each concanavalin A receptor is able to perturb about 685 molecules of dimyristoylphosphatidylcholine, withdrawing them from the main phase transition. The cooperativity of phospholipid melting is profoundly disrupted by small amounts of glycoprotein, with the cooperative unit dropping to less than half its initial values at a protein/lipid mole ratio of 1:3800. A break occurs in the ΔH curve as the protein/lipid mole ratio is increased above 1:1160, and ΔH then increases linearly with increasing amounts of concanavalin A receptor in the bilayer. This phenomenon may be interpreted in terms of protein-protein aggregation which occurs in the phospholipid bilayer above a certain critical mole fraction of concanavalin A receptor, resulting in perturbed phospholipids being returned to the phase transition. In addition, the hydrophilic domains of the glycoprotein may exist in two different conformations depending on the protein concentration in the bilayer, and these may differ in their ability to interact with phospholipid headgroups at the membrane surface.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

Production and Characterization of a Pressure-induced Gel from Freeze-concentrated Milk

A process was developed to produce a characteristic milk gel. Raw and market milk samples were freeze-concentrated using bacterial ice nuclei. The concentrates were kept at 5°C and compressed at 300–600 MPa for 5 min. The combination of the freeze concentration and the pressurization gave a milk gel without adding any gelling agents. The addition of sugar at 10% to the concentrated milk improved its gel strength and viscoelasticity. The gel was characterized by a phase transition at about 62–75°C.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.