Hydrogenation of Triton X-100 eliminates its fluorescence and ultraviolet light absorption while preserving its detergent properties

The ultraviolet-light absorption and fluorescence of Triton X-100 were virtually eliminated by hydrogenation to its reduced cyclohexyl analog, RTX-100. The critical micelle concentration of RTX-100 was 12% higher than that of Triton X-100. RTX-100 and Triton X-100 were quite similar in their abilities to extract proteins from human erythrocyte membranes.     

Triton X-100 alters the resistance level of methicillin-resistant Staphylococcus aureus to oxacillin

Abstract We used population analysis to examine the effects of Triton X-100 on the level of resistance to oxacillin of 18 methicillin-resistant Staphylococcus aureus . In the presence of 0.02% Triton X-100, 17 formerly methicillin-resistiant strains exhibited enhanced sensitivity to oxacillin. One homogeneous isolate, KSAF1 was barely affected by the Triton X-100. Sensitivities of lysostaphin, 51 kDa N -acetylglucosaminidase and 62 kDa N -acetylmuramoyl-l-alanine amidase to heat-inactivated cells were not affected when the bacteria were grown in 0.02% Triton X-100. Our data, together with those of a previous study, suggested that Triton X-100 alters the resistance level of methicillin-resistant S. aureus by influencing a factor(s) other than PBPs, bacteriolytic enzymes, or femAB products.     

Triton X—100加KI对燕麦根细胞质膜ATP酶的溶解

在酸性条件下,1％ Triton X—100加 0.25mol/L KI能有效地溶解燕麦根细胞质膜ATP酶。溶解的ATP酶水解ATP的最适pH在6.5左右,酶活性受到Na_3VO_4和DES的强烈抑制,而不受Na_2MoO_4和NaN_3的抑制。溶解的酶液经透析后,K~ —ATP酶活性占Mg~(2 ),KCl—ATP酶活性的85％。     

Spectroscopic studies on the denaturation of papain solubilized and Triton X-100-solubilized glucoamylase from rabbit small intestine

Intestinal brush border proteins consist of an enzymatically active hydrophilic moiety attached to a hydrophobic tail. Papain dissociates the hydrophilic part by cleaving off the hydrophobic tail, whereas the detergentTriton X-100 solubilizes the whole molecule. Denaturation by 8 M urea or 4 M guanidinium chloride does not alter the structure of the papain-solubilized enzyme. An appreciable alteration of the structure of detergent-solubilized enzyme was observed on denaturation. The difference spectra of Triton X-100 (1%)—solubilized enzyme and its urea denatured form shifts and intensifies, with increase in the concentration of the denaturant with an isobestic point at 252 nm. A new band at 280 nm also appears at 4 M urea concentration. Papain-solubilized glucoamylase has an ∞ -helical conformation in solution unlike the detergentsolubilized fraction. An elongated structure for the papain solubilized enzyme is inferred from the urea denaturation studies and from molecular weight determinations.     

Energy transfer in the light-harvesting complex II of Dunaliella tertiolecta is unusually sensitive to Triton X-100

Triton X-100, a detergent commonly used to solubilize higher plant thylakoid membranes, was found to be deleterious to Dunaliella LHC II. It disrupted the transfer of excitation energy from chlorophyll b to chlorophyll a. Based on analysis of pigments and immunoassays of LHC II apoproteins from sucrose density gradient fractions, Triton X-100 caused aggregation of the complex, but apparently did not remove chlorophyll b from the apoprotein. Following solubilization with Triton X-100 only CPI could be resolved by electrophoresis. In contrast, solubilization of Dunaliella thylakoids with octyl--D-glucopyranoside preserved energy transfer from chlorophyll b to chlorophyll a. This detergent also effectively prevented aggregation on sucrose gradients and preserved CPI oligomers, as well as LHCP1 and LHCP3 on non-denaturing gels. Solubilization with Deriphat gave similar results. We propose that room temperature fluorescence excitation and emission spectroscopy be used in conjunction with other biophysical and biochemical probes to establish the effects of detergents on the integrity of light harvesting chlorophyll protein complexes. Methods used here may be applicable to other chlorophytes which prove refractory to protocols developed for higher plants.Abbreviations LHC II light harvesting chlorophyll protein complex associated with photosystem II - LHCP1 and LHCP3 monomeric and oligomeric forms of LHC II, respectively, observed on non-denaturing gels - LiDS lithium dodecylsulphate - PMSF phenylmethylsulfonyl fluoride     

Temperature-dependence of the solubilization of dipalmitoylphosphatidylcholine (DPPC) by the non-ionic surfactant Triton X-100, kinetic and structural aspects

Most of the studies on the solubilization of model membranes conducted thus far involved model membranes made of liquid-crystalline phospholipids. Relatively little is known on the influence of temperature and of the phase of the lipid bilayers on their solubilization by detergents. The aim of the present study was to gain knowledge about the temperature and phase dependence of the solubilization of phospholipid bilayers by the non-ionic detergent Triton X-100 (TR). Detailed investigation of the kinetics of the solubilization of dipalmitoylphosphatidylcholine (DPPC), as well as of palmitoyloleoylphosphatidylcholine (POPC) by TR at different temperatures reveals that: (i) solubilization of DPPC is a relatively slow process, especially below Tm. This means that in order to prevent misleading conclusions it is important to monitor the solubilization after a steady state is established. (ii) Both the steady state structure and size of DPPC/TR aggregates and the kinetics of solubilization depend on temperature. (iii) The TR concentration required for solubilization of POPC bilayers is an increasing function of temperature, although no phase change of bilayers occurs in the studied temperature range. (iv) Detailed studies of the temperature-induced changes of the aggregates present in DPPC/TR or POPC/TR mixtures suggest that the state of aggregation at any temperature above 23 degrees C represents equilibrium. By contrast, for DPPC/TR mixtures at 4 degrees C all the processes are very slow, which complicates the interpretation of results obtained through the common practice of studying "rafts" by investigating detergent-resistant membranes.     

The fluorescence lifetime and quantum yield of chlorophyll a in Triton X-100 solutions

The fluorescence of chlorophyll (Chl) a in 0.007–0.1% Triton X-100 was investigated by a phase-shift technique. The Chl a concentrations varied from 0.7 to 25 μM. Parallel measurements of fluorescence lifetime (τ) and quantum yield (ψ) were made. It was concluded that homogeneous energy transfer takes place at detergent concentrations above 0.025%: (i) the transfer between uniform molecules of the pigment solubilized in Triton X-100 micelles, when τ and ψ are constant; (ii) the transfer towards the quenching centers, resulting in a proportional decrease in τ and ψ. At a Triton X-100 concentration of about 0.025% the Chl a emission becomes heterogeneous. It is evident from the disproportional decrease in τ and ψ (greater in ψ than in τ) and also from the rise of the fluorescence at 730–750 nm. As the Triton X-100 concentration becomes lower than the critical one (0.021%), the number of micelles drops abruptly and Chl a forms colloid particles in the aqueous medium. This manifests itself as a decrease in τ and as a certain stabilization of ψ. Having analyzed the complex pattern of the τ/ψ ratio, we concluded that under these conditions more than 90% of Chl a is in a weakly fluorescent form (τ < 30 ps) and about 1% is in an aggregated state fluorescing at 732 nm with τ about 0.7 ns.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in Triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties

1. A method for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography.2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with M_r of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 μmol of cytochrome b, 4 μmol of cytochrome c₁ 7–8 μmol of nonheme iron, corresponding to 3.5–4 μmol of the Rieske iron-sulfur protein, less than 1.0 μmol of ubiquinone and about 60 μmol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2 g of Triton X-100 per g protein.3. Cytochrome b exhibits an α-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. ?10 and +100 mV, at pH 7.0. The absorbance maximum of cytochrome c₁ lies at 553 nm and its half-reduction potential amounts to +250 mV.4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20°C.5. A dissociation constant K′_s of 5.5 mM has been determined for the Triton-solubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas.6. A K′_m value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.     

DMSO、Tween-20和Triton X-100对野葛悬浮细胞异黄酮生产的影响

二甲基亚砜(DMSO)、Tween-20和Triton X-100可以增大悬浮培养细胞的细胞膜和液泡膜的透性,促进细胞内次生代谢物的释放,从而影响这些次生代谢产物的产量。为了提高野葛悬浮细胞中异黄酮类化合物的产量,以1％、3％和5％的DMSO、Tween-20和Triton X-100分别处理野葛叶悬浮细胞,结果显示,Tween-20和Triton X-100皆明显促进细胞生物量和葛根素和异黄酮化合物的释放。5％的Triton X-100处理3d,促进细胞产生总异黄酮化合物,增产率达40．6％。     

Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins

Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC–MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC–MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and elongation, as well as steroid synthesis. In addition, transport proteins including 24 members of the Rab family of GTPases were identified. Comparison of this dataset with the current mouse liver microsome proteome contributes an additional 648 protein identifications, of which 50% (326/648) contain at least one TMD.     

The phospholipid fatty acids of Porphyridium purpureum cultured in the presence of triton x-100 and sodium desoxycholate

The phospholipid fatty acid composition of Porphyridium purpureum grown on a solid medium was studied in the presence of Triton X-100 (TX) and sodium desoxycholate (SDC). The most common fatty acids in PC and PE were palmitic (16:0), stearic (18:0), linoleic (18:2ω6), arachidonic (20:4ω6) and eicosapentaenoic (20:5ω3) acids, 20:4ω6 being very abundant. In PG the most common acids were 16:0, trans-hexaenoic acid (tr16:1ω3), oleic acid (18:1) and 20:4ω6. Both detergents caused an increase in the saturation of PC and, to a lesser extent, of PE. The relative amounts of short chain fatty acids increased. Both detergents increased the amounts of 16:0 and, correspondingly, decreased the amounts of 20:4ω6. In PG the amounts of both 16:0 and tr 16:1ω3 increased and the amounts of 18:0, 18:2ω6 and 20:4ω6 decreased in the presence of detergents. The changes were always greatest at the concentrations of 5–10 ppm TX or SDC. At 20 ppm the fatty acid compositions, especially with SDC, were very similar to the controls, which suggests a change in the detergent effect between 10–20 ppm. The normal PC/PE ratio was 5.6 and the (PC+ PE)/PG ratio 39.0. Both detergents caused a marked decrease in these ratios. Because the detergent effects are not linear, it seems that even very low detergent concentrations have an important influence on algae in polluted waters.     

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b-559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b-563, cytochrome f, phylloquinone, α-tocopherol or α-tocopherylquinone.     

Triton x-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABAA receptors in Xenopus oocytes

Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na⁺ and L-type Ca²⁺ channels and GABA_A receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA_A receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA_A α₁β₃γ_2S receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 μM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA_A receptor currents and suppressed allosteric modulation by flunitrazepam at α₁β₃γ_2S receptors. All effects were independent of the presence of a γ_2S subunit in the GABA_A receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA_A receptor through changes in lipid bilayer elasticity.     

Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana

Thylakoids of the diatom Cyclotella meneghiniana were separated by discontinuous gradient centrifugation into photosystem (PS) I, PSII, and fucoxanthin-chlorophyll protein (FCP) fractions. FCPs are homologue to light harvesting complexes of higher plants with similar function in e.g. brown algae and diatoms. Still, it is unclear if FCP complexes are specifically associated with either PSI or PSII, or if FCP complexes function as one antenna for both photosystems. However, a trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have been described for C. meneghiniana, already. In this study, biochemical and spectroscopical evidences are provided that reveal a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PSII associated Fcp antenna resembles FCPa since it contains Fcp2 and Fcp6, at least three different Fcp polypeptides are associated with PSI. By re-solubilisation and a further purification step Fcp polypeptides were partially removed from PSI and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby a protein related to Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PSI, whereas presumably Fcp5, a subunit of the FCPb complex, is only loosely bound to the PSI core. Thus, an association of FCPb and PSI is assumed.     

S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature)

The S100 protein family is the largest subgroup within the superfamily of proteins carrying the Ca2+-binding EF-hand motif. Despite their small molecular size and their conserved functional domain of two distinct EF-hands, S100 proteins developed a plethora of tissue-specific intra- and extracellular functions. Accordingly, various diseases such as cardiomyopathies, neurodegenerative and inflammatory disorders, and cancer are associated with altered S100 protein levels. Here, we review the different S100 protein functions and related diseases from an evolutionary point of view. We analyzed the structural variations, which are the basis of functional diversification, as well as the genomic organization of the S100 family in human and compared it with the S100 repertoires in mouse and rat. S100 genes and proteins are highly conserved between the different mammalian species. Moreover, we identified evolutionary related subgroups of S100 proteins within the three species, which share functional similarity and form subclusters on the genomic level. The available S100-specific mouse models are summarized and the consequences of our results are discussed with regard to the use of genetically engineered mice as human disease models. An update of the S100 nomenclature is included, because some of the recently identified S100 genes and pseudogenes had to be renamed.     

Decreased intracellular GABA levels contribute to spinal cord stimulation-induced analgesia in rats suffering from painful peripheral neuropathy: the role of KCC2 and GABA(A) receptor-mediated inhibition

Elevated spinal extracellular γ-aminobutyric acid (GABA) levels have been described during spinal cord stimulation (SCS)-induced analgesia in experimental chronic peripheral neuropathy. Interestingly, these increased GABA levels strongly exceeded the time frame of SCS-induced analgesia. In line with the former, pharmacologically-enhanced extracellular GABA levels by GABA_B receptor agonists in combination with SCS in non-responders to SCS solely could convert these non-responders into responders. However, similar treatment with GABA_A receptor agonists and SCS is known to be less efficient. Since K⁺ Cl⁻ cotransporter 2 (KCC2) functionality strongly determines proper GABA_A receptor-mediated inhibition, both decreased numbers of GABA_A receptors as well as reduced KCC2 protein expression might play a pivotal role in this loss of GABA_A receptor-mediated inhibition in non-responders. Here, we explored the mechanisms underlying both changes in extracellular GABA levels and impaired GABA_A receptor-mediated inhibition after 30 min of SCS in rats suffering from partial sciatic nerve ligation (PSNL). Immediately after cessation of SCS, a decreased spinal intracellular dorsal horn GABA-immunoreactivity was observed in responders when compared to non-responders or sham SCS rats. One hour later however, GABA-immunoreactivity was already increased to similar levels as those observed in non-responder or sham SCS rats. These changes did not coincide with alterations in the number of GABA-immunoreactive cells. C-Fos/GABA double-fluorescence clearly confirmed a SCS-induced activation of GABA-immunoreactive cells in responders immediately after SCS. Differences in spinal dorsal horn GABA_A receptor-immunoreactivity and KCC2 protein levels were absent between all SCS groups. However, KCC2 protein levels were significantly decreased compared to sham PSNL animals. In conclusion, reduced intracellular GABA levels are only present during the time frame of SCS in responders and strongly point to a SCS-mediated on/off GABAergic release mechanism. Furthermore, a KCC2-dependent impaired GABA_A receptor-mediated inhibition seems to be present both in responders and non-responders to SCS due to similar KCC2 and GABA_A receptor levels.     

First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui

A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far–UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25–0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.