Triton Treatment Research Articles

Temperature-independent binding of[ 3H](±)-3-(2-car☐ypiperazin-4-yl)propyl-1-phosphonic acid in brain synaptic membranes treated by Triton X-100

Specific binding of[ 3H](±)-3-(2-car☐ypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a highly selective antagonist forN-methyl- d-aspartic acid (NMDA) receptors, was examined in brain synaptic membranes treated with Triton X-100 by using a filtration assay method. Elevation of incubation temperature from 2 to 30 °C markedly diminished the binding. The binding reached a plateau within 5 min after the initiation of incubation at 2 °C, while the time required to attain an equilibrium was 1 min at 30 °C. The binding at 2 °C was rapidly dissociated by the addition of an excess of unlabeled CPP, NMDA and l-glutamic acid ( l-Glu). The binding was also saturable with increasing concentrations of the ligand and displaced by various amino acids structurally related to l-Glu in a stereospecific manner. Competitive but not noncompetitive antagonists for the NMDA receptors invariably inhibited the binding. However, the binding was not prominently affected by agonists for the other subclasses of the brain excitatory amino acid receptors. Both reduced and oxidized forms of glutathione significantly displaced the binding. Scatchard analysis revealed that Triton treatment increased the affinity and density of binding sites which consisted of a single component. Among some endogenous tryptophan metabolites, kynurenic, anthranilic and quinolinic acids inhibited the binding. These results suggest that a filtration assay method is also useful to detect the binding of NMDA receptors in the brain.

Effects of acromelic acid A on the binding of [ 3H]glutamic acid and [ 3H]kainic acid to synaptic membranes and on the depolarization at the frog spinal cord

Triton treatment of synaptic membranes from the frog spinal cord enhanced the specific binding of [ 3H]glutamic acid compared with non-treated fresh and frozen ones, but not that of [ 3H]kainic acid. Acromelic acid A specifically inhibited the binding of [ 3H]kainic acid, and was approximately 100 times more potent than kainic acid. Acromelic acid A and excitatory amino acids caused a depolarization in the ventral root of the frog spinal cord in a dose-dependent manner, and the effect of acromelic acid A was much superior to that of kainic acid or domoic acid. Acromelic acid A is one of the most potent kainic acid agonist at the frog spinal cord.

Labeling of NMDA receptor channels by [ 3H]MK-801 in brain synaptic membranes treated with Triton X-100

Binding activity of [ 3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was examined by using rat brain synaptic membranes treated with Triton X-100. This compound is known as a non-competitive antagonist for one subclass of the central excitatory amino acid receptors, N-methyl- d-aspartic acid (NMDA)-sensitive receptors. Triton treatment completely abolished the temperature-dependent portion of the binding activity, with a concomitant reduction of membranous protein content. Addition of l-glutamic acid (Glu), however, markedly potentiated the activity at concentrations higher than 10 nM in a temperature-dependent manner. Similarly significant potentiation was induced by structurally related amino acids as well as agonists for the NMDA-sensitive receptors, but not by agonists for the other subclasses. The rank order of this stimulatory potency was well consistent with that of the displacing activity of these Glu analogues on NMDA-sensitive [ 3H]Glu binding. Competitive NMDA antagonists, such as (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid and d-2-amino-5-phosphonovaleric acid, were all effective in preventing the potentiation of [ 3H]MK-801 binding activity by l-Glu. The latter Glu-dependent activity was additionally enhanced by glycine and its analogues at concentrations above 10 nM in a temperature- and Glu-dependent fashion. Additional enhancement by glycine was also antagonized by competitive NMDA antagonists, but not by a classical glycine antagonist strychnine. These results suggest that Triton X-100-treated membranes are suitable for the study of the interaction of the non-competitive antagonists with NMDA receptor channels, and may be superior to non-detergent-treated membranes in terms of freedom from the confounding effects of endogenous amino acids.

Cortical benzodiazepine receptor binding in a rabbit model of hepatic encephalopathy: The effect of Triton X-100 on receptor solubilization

Increased benzodiazepine (BZ) receptor density has been reported in brains of rabbits with hepatic encephalopathy (HE) due to galactosamine (GalN)-induced fulminant hepatic failure (FHF). These data were generated using detergent-Triton X-100-treated neural membranes. While performing further studies it was noted that the increase in BZ receptor density was not demonstrable when Triton X-100 preparation was not employed. Accordingly the binding of [3H] flunitrazepam, a BZ ligand, to neural membranes from cortices of normal rabbits and rabbits with HE due to (GalN)-induced FHF was studied with and without detergent preparation. Scatchard plot analysis of the binding data indicated that when no detergent was employed, the apparent affinity and density of BZ receptors were similar for control membranes and membranes from animals in HE. BZ receptors from animals in HE were shown to be more resistant to solubilization by Triton than control membranes. These findings (a) afford a potential explanation for the apparent increase in density of BZ receptors in this model when Triton treatment of neural membranes is utilized and (b) suggest that recent evidence for increased GABAergic tone in the syndrome of HE is not dependent on an increased density of BZ receptors.

Enhanced number of actin binding sites on plasma membranes of polyoma virus-transformed fibroblasts

Plasma membranes, isolated from normal (C13) and polyoma virus-transformed (J1) cultured BHK cells were incubated with G-actin under polymerizing conditions, followed by a low-speed centrifugation. The amount of actin attached to the pelleted BHK-J1 plasma membranes was at least twice that on BHK-C13 membranes, indicating a greater number of actin attachment sites on the former. This result was confirmed by the observation that the plasma membranes from the transformed cells were also more active in nucleating polymerization of pyrene-labelled actin. Most of the actin attachment sites could be solubilized by Triton or low-salt extraction treatment.

A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V.

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.

Polygonal networks, “geodomes”, of adult rat hepatocytes in primary culture

Polygonal networks, “geodomes”, in cultured hepatocytes of adult rats were examined by both light and electron microscopy. On light microscopical examinations of specimens stained with Coomassie blue after the treatment with Triton X-100, the networks were detected 5 days after culture, which consisted of triangles arranged mainly in hexagonal patterns. They surrounded main cell body, looking like a headband, or were occasionally situated over nuclei, looking like a geodesic dome. Scanning electron microscopical observations after Triton treatment revealed that these structures were located underneath surface membrane. Transmission electron microscopical investigations revealed that the connecting fibers of networks consisted of microfilaments which radiated in a compact bundle from electron-dense vertices.

The effect of Triton X-100 on bovine brain synaptic membranes.

Bovine brain synaptic membranes which were frozen and then extensively washed showed low affinity [3H]muscimol binding. These membranes contained GABA and calmodulin, apparently tightly bound within the membrane fraction. Membranes which were additionally treated with the detergent Triton X-100 showed high affinity [3H]muscimol binding. These membranes did not appear to contain GABA or calmodulin. Transmission electron microscopy studies demonstrated that the washed membrane fraction contained many synaptosomal and vesicular structures. Triton treatment led to the extensive rupture of these structures. These studies explain the well-reported findings of tightly bound GABA and calmodulin in brain membrane fractions, as being due to the entrapment of these compounds inside sealed membrane-bound structures which are still present after a freeze-thaw and extensive wash treatment, their complete removal requiring Triton-treatment to rupture the vesicles.

Selenium-mediated biochemical changes in Japanese quails

The tissue uptake and distribution of injected [(75)Se]-sodium selenite as a variance with time and as influenced by dietary selenium status was followed in the tissues of Japanese quails,Coturnix coturnix japonica. Quails maintained on a low selenium semipurified (basal) diet and basal diets supplemented with 0.2 and 2.0 ppm selenium as sodium selenite were injected intraperitonially with(75)Se as sodium selenite (2.8 microcuries). The injected(75)Se was monitored in blood, liver, kidney, heart, and testis at 24, 72, and 144 h after injection. Maximal uptake of the injected(75)Se was observed in tissues of quails maintained on basal diet. The uptake of(75)Se in tissues in general was determined by the dietary Se status. Among the organs studied, kidney had the maximal level of(75)Se, 0.2 ppm (μg/g wet tissue) followed by liver, testis, and heart, but testis had the maximal level when the level per milligram of protein was considered, about 3.0 ng/mg protein, followed by liver, kidney, and heart. About 10-20% of the tissue(75)Se was located in the mitochondria and 50-60% in the post-mitochondrial supernatant fractions in all dietary Se levels. Significant incorporation of(75)Se in the mitochondrial membrane was observed. The percent distribution ratio between the membrane and matrix fractions of the mitochondria remained constant at all dietary Se levels which, in liver was 65∶35, in kidney 55∶45, and in testis 75∶25. However, in heart mitochondria, the distribution of(75)Se between membrane and matrix varied with dietary Se status, the ratio being 82∶18 in the basal group, and 72∶28 and 41∶59 in the 0.2 and 2.0 ppm Se-supplemented groups, respectively. This is indicative of a preferential uptake of(75)Se in the mitochondrial membrane in conditions of deficiency. About 40-60% of the mitochondrial membrane-associated(75)Se was released upon Triton treatment in all the organs. Of the membrane-bound(75)Se, about 10-15% was acid-labile in liver and kidney and 25% in the heart tissue. Possibilities of tissue specific roles, especially in the heart mitochondrial membrane-related processes, are indicated for selenium.

Blockade of intestinal lipoprotein clearance in rabbits injected with Triton WR 1339-ethyl oleate.

Although Triton WR 1339 has been used to block triglyceride or cholesterol removal from plasma, no data are available on the extent to which Triton WR 1339 administered to rabbits blocks clearance of newly absorbed dietary lipids. In the present study, we have measured the efficiency of this blockade during a 24-hr interval. After the Triton WR 1339 administration, plasma Sf greater than 400 and d less than 1.019 g/ml lipoprotein lipid concentrations increased greatly, but the concentration of d greater than 1.019 g/ml lipids decreased. In the rabbits fed 0.5% cholesterol for 1 week, the increase in d less than 1.019 g/ml and the decrease in 1.019 less than d less than 1.063 g/ml lipoprotein fractions 24 hr after the Triton WR 1339 injection were much greater than in the chow-fed Tritonized rabbits. After the Triton treatment, 50% of intravenously injected LDL-125I-labeled apoB disappeared in 24 hr, but little or no apoB appeared in other lipoprotein fractions and no VLDL apoB was converted to LDL. Labeled cholesterol and retinol were fed to rabbits and 24-hr increments in plasma cholesteryl- and retinyl-ester label and mass were measured. In chow-fed Tritonized rabbits about one-half of the absorbed oral doses of both labeled lipids was recovered in plasma, indicating that Triton WR 1339 does not completely inhibit the clearance of intestinal lipoproteins. When rabbits were injected with Triton and an ethyl oleate emulsion, the blockade of dietary lipid removal from plasma was substantially improved and chylomicron cholesterol uptake by extra-hepatic tissues was completely abolished.

The effects of detergent on the contractility and ultrastructure of frog skeletal muscle.

The contractility of detergent-treated frog skeletal muscle and its ultrastructure were studied. Triton X-100 and Brij-58 were used as nonionic surface active substances. The concentrations of the detergent solutions were adjusted so that the trains of the twitch tensions would continue for about 10 min. The duration of the twitch tensions was about 10 min when the muscles were soaked in 0.1 mM (0.006%) Triton or in 2.5 mM (0.280%) Brij solution. The twitch tensions did not recover upon returning the muscles to the normal Ringer solution after treatment of the detergents. Rapid cooling of muscle that had been soaked in a 1.0 mM caffeine Ringer solution for 10 min provoked a marked contracture (RCC) in 0.1-0.2 mM Triton-treated preparation. No such contracture was obtained in 1.0-2.5 mM Brij-treated preparation. In ultrastructural findings of the Triton-treated muscle, some structural changes were observed in the transverse tubule and the junctional gap, but not in the terminal cisternae. Brij-treated muscle showed damage of each of these membranes, including the terminal cisternae. These findings suggest that the two detergents have different surface activities on the internal membrane system of the skeletal muscle. The biological activities of the membrane remained even after the treatment of Triton with a suitable concentration.

Dynamic aspects of the contractile system in Physarum Plasmodium: II. Contractility of triton cell models in accordance with the contraction and relaxation phases of the plasmodia

AbstractThe contractility of Physarum plasmodium was investigated using cell models that were prepared by treating thin‐spread plasmodia with ice‐cold 0.2% Triton X‐100. Cell models obtained from the anterior regions of the thin‐spread plasmodia in the contraction phase retained many birefringent cytoplasmic fibrils. The fibrils vigorously contracted on addition of ATP, inducing simultaneous contraction of the whole cell models. In contrast, cell models prepared from the anterior regions in the relaxation phase scarcely contained the birefringent fibrils and exhibited only weak contractility on addition of ATP. The posterior regions of the thinspread plasmodia, which were composed of ramified plasmodial strands, always retained many fibrils when treated with the Triton solution and showed intensive contraction on addition of ATP.SDS‐polyacrylamide gel electrophoresis showed that the model was enriched for actin and myosin. About 40% of the actin was extracted from the plasmodium by the Triton treatment, while scarcely any myosin was extracted.Fragmin, a F‐actin‐fragmenting factor, caused the birefringent fibrils to diminish in the presence of Ca2+, but more than 30 minutes was required for their complete disappearance. The birefringent fibrils weakened by 30‐minute fragmin treatment disappeared immediately on addition of ATP or AMP‐PNP.

A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays.

Two commonly used procedures for removing endogenous GABA from brain homogenates were evaluated by measuring residual GABA using high performance liquid chromatography (HPLC). The effect of these treatments on [3H]muscimol binding to the GABA receptor was also determined. Membranes subjected to osmotic lysing and eight washes with Tris-citrate buffer contained significant quantities of residual GABA whereas lysing and incubation with Triton X-100 followed by three buffer washes resulted in GABA levels below the limits of detection. The apparent affinity for [3H]muscimol was significantly higher in the Triton X-100 treated membranes and this was probably a result of the lower amount of GABA present in these membranes. The effect of Triton treatment or buffer washing on residual levels of glutamate, glutamine, aspartate, and taurine were also determined.

Characterization of beta-glucosidase as a peripheral enzyme of lysosomal membranes from mouse liver and purification.

Lysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of beta-glucosidase [EC 3.2.1.45] in lysosomes was associated with the membrane fractions, whereas 96% of beta-glucuronidase [EC 3.2.1.31] was recovered in the soluble fractions of lysosomes. beta-glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that beta-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkali-treated enzyme. These results suggested that beta-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. beta-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.

Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: protein crosslinking studies.

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.

Effect of Triton X-100 on insulin and epidermal growth factor receptor binding and autophosphorylation in Golgi fractions and partially purified receptors from rat liver.

Triton X-100 strongly affects the receptor binding and autophosphorylation of insulin and epidermal growth factor (EGF) in rat liver Golgi fractions and partially purified microsomal receptors. At concentration 0.05% Triton X-100 decreased the insulin receptor binding by 15% and the EGF receptor binding by 70% as compared to controls. In contrast, 0.05% Triton X-100 increased insulin-stimulated receptor autophosphorylation by more than 370% as compared to 87% in the control. Similarly, the same concentration of Triton X-100 increased the EGF-stimulated receptor phosphorylation by 65% as compared to 14% in the control. EGF receptor binding was more sensitive to the treatment of Triton. At Triton concentrations 0.2% or more, the EGF receptor binding was totally abolished while the insulin receptor binding was decreased by 50%. On the other hand, the activity of ligand-stimulated receptor phosphorylation of both insulin and EGF receptors was only slightly decreased in the presence of 0.2% Triton.

Immobilization of yeast cells with high β-galactosidase activity

K. lactis batch growth conditins were optimized for high B-galactosidase content using a modified inorganic salt medium with 1 % lactose at 300C. The pH was found to be a suitable indicator for the optimum harvest time. Cell wall permeabilization by Triton yielded β-galactosidase activity of up to 966 LAU (g cell)-1. The effect of Triton treatment was documented by SEM pictures of the cells. The cells were immobilized in polyurethane foam and used in a continuous plug-flow reactor with a half-life time of approximately 3 days. It was shown that both the free as well as the immobilized cells utilize glucose in a follow-up reaction and, therefore, kinetic measurements based on glucose concentrations alone are not sufficient to evaluate true kinetic parameters.

Effect of intrabiliary administration of triton X-100 on biliary excretory function in the rat

Intrabiliary administration of Triton X-100 is of interest in producing effects on biliary tree permeability and canalicular biliary excretory function. Treatment with 0.4% Triton (40 μl) was shown to increase the biliary excretion of intraportably administered [ 3H]sucrose. It also decreased recovery of [ 3H]sucrose given into the biliary tree. Thus, we concluded that Triton treatment increased biliary tree permeability. Using a different set of marker compounds, canalicular transport of bromphenol blue, [ 14C]morphine glucuronide and [ 3H]ouabain was found to be decreased. The fact that [ 3H] taurocholate excretion into bile was not affected whereas that of [ 3H]ouabain was lends support to the concept that taurocholate and ouabain are not transported by a common pathway.

Tegumental disruption with Triton X-100 and its effects on longitudinal muscle function in male Schistosoma mansoni.

Longitudinal muscles in adult male Schistosoma mansoni appear to remain in almost all respects functional after tegumental disruption by Triton X-100. Responses to high K+, ouabain, low temperature, praziquantel and neurotransmitters are still present. Muscle membrane potentials remain near those of control animals and lanthanum nitrate is still excluded from the muscle after the Triton treatment. Although muscle contractility remains after tegumental disruption, sodium and calcium levels, as measured by plasma emission spectroscopy, increase by 20-30% while potassium and magnesium levels decrease by 15%. 45Ca2+ accumulation is double that of control animals. Upon exposure to a bathing medium lacking Ca2+, Triton-treated animals lose responsiveness to high K+, ouabain, low temperature and praziquantel more rapidly than do control animals. D-600, which effectively blocks the high K+ response in control animals, has no measurable effect on the high K+ response in Triton-treated animals, suggesting that voltage-dependent Ca2+ channels are present in the tegument.

Studies on fat digestion, absorption, and transport in the suckling rat. IV. In vivo rates of triacylglycerol secretion by intestine and liver.

The rate of triacylglycerol entry into plasma in 14- to 15-day-old suckling rats was measured using the Triton WR1339 method. When the pups were left with the mother and allowed to nurse continually, the rate was 31.8 +/- 2.3 mumol/hr per rat. If the pups were removed from the mother with full stomachs and kept in a warm box at 30-32 degrees C, the rate was 12.5 +/- 1.5 mumol/hr per rat. The rate of triacylglycerol output from the intestine measured in lymph duct-cannulated animals (kept in a warm box at 30-32 degrees C) was 14.9 +/- 2.7 mumol/hr per rat. Triton treatment of lymph duct-cannulated animals caused only a small increase in plasma triacylglycerol concentration (less than 2 mumol/hr per rat). These data suggest that nearly 100% of plasma triacylglycerol arises from intestinal input in the suckling rat.