Effects of detergents and choline-containing phospholipids on human spleen glucocerebrosidase.

1. Glucocerebrosidase, extracted from human spleen lysosomal membrane by sodium cholate and recovered in a high speed centrifugation supernatant, aggregated following removal of the detergent. 2. Re-solubilization of the enzymatic activity from the aggregate was achieved by treatment with the non-ionic detergents Triton X-100 and Tween 20. The anionic detergents sodium cholate and sodium taurocholate and the cationic detergents cetyltrimethylammonium bromide and cetylpyridinium chloride were also effective. The solubilizing capacity of the anionic detergents was smaller than that of the nonionic detergents. Quantitative evaluation of the solubilizing capacity of the cationic detergents was not feasible because of their being potent inhibitors of glucocerebrosidase activity. 3. Treatment of the enzyme aggregate with acetone rendered it buffer-soluble. 4. In addition to the above cationic detergents some choline-containing and highly hydrophobic phospholipids were found to inhibit the glucocerebrosidase activity.     

Effects of detergents on the West Nile virus protease activity

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at ?0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC₅₀ value of 5.7 ± 2.5 μM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.     

Sensitivity of Escherichia coli cell membranes to various classes of detergents

The mechanisms of interaction between non-ionic or cationic surfactants with Escherichia coli K-12 cell membranes were studied using an approach based on the registration of changes in the membrane permeability to ethidium bromide, a fluorescent dye for nucleic acids. Triton X-100, a non-ionic detergent, was shown to exert no effect on the permeability of intact cell membranes. Triton X-100 interacted with the bacteria only after treatment with EDTA, a complexing agent for bivalent cations. Cetyltrimethyl ammonium bromide increased the permeability to ethidium bromide and the action of this cationic detergent did not require the pretreatment with the complexing agent. SDS, an anionic detergent, damaged E. coli K-12 and this could be registered by the lowering of intensity of light scattering by the bacterial suspension. The surface charge of E. coli K-12 cells was shown to influence the interaction of ionic detergents with bacterial cell membranes. Its variation by changing the pH of the incubation medium did not make E. coli K-12 sensitive to Triton X-100.     

The effects of cations on the structure and photochemistry of the photosystem II core complex

We have studied the effects of cations and detergents on the structure (molecular weight) and photochemistry of Triton X-100 Photosystem II subchloroplast particles (TSF-IIa). The effect of Mg²⁺ ions on activity depended on the Triton X-100 content of the preparation. If the residual Triton X-100 was not removed prior to assay, MgCl₂ increased the rate of electron transport, acting at a site on the reducing side of Photosystem II. Lowering the pH also increased the rate of electron transport. If the Triton X-100 was removed from the particles, both MgCl₂ and NaCl caused a decrease in the rate of electron transport. Addition of Triton X-100 caused a reversible decrease in the number of active Photosystem II reaction centers. Both cations and Triton X-100 had a profound effect on the molecular weight of the Photosystem II particles as determined by gel filtration. At 20 °C, addition of 0.05% Triton X-100 decreased the molecular weight from a high value (≥800,000) to 250,000. At 4 °C, addition of 1 mm MgCl₂ or 100 mm NaCl increased the molecular weight of the complex. In the absence of these salts 67% of the protein eluted with a molecular weight of 460,000 (the rest was >800,000-in the void volume). In the presence of these salts all of the material had a molecular weight of ≥800,000. A similar effect was observed when the pH was lowered from 8 to 6. Further work is needed to determine whether there is a correlation between the changes in molecular weight and activity.     

Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.     

Utilization of zeolite Y in the removal of anionic,cationic and nonionic detergents during purification of proteins

Summary Hydrophobie zeolite Y was used to adsorb detergents from protein solutions and within one minute the commonly used detergents sodium dodecyl sulfate, cetyl trimethyl ammonium bromide, and Triton X-100 at concentrations of 10 mg/ml were adsorbed to a level below their critical micelle concentrations. From the detergent depleted solutions 77 to 85 % of the proteins were recovered; the lower value was obtained with protein concentration below one mg/ml.     

Coupled oxidation of carotene by lipoxygenase requires two isoenzymes.

The esterase activity of rat urinary kallikrein is increased up to fourfold by the anionic detergent, deoxycholate and the nonionic detergents, Triton X-100, Lubrol PX, and Brij 58. The cationic detergents, benzyltriphenylphosphonium chloride and cetyltri-methylammonium bromide, inhibit kallikrein activity. Certain trypsin inhibitors stimulate kallikrein activity but this stimulation is not observed when kallikrein is preincubated with deoxycholate. In addition, deoxycholate weakens the inhibition of kallikrein activity by Trasylol. Deoxycholate-induced conformational changes of kallikrein are noted by a change in circular dichroism spectra in the far and near ultraviolet region. A maximal change of ellipticity at 275 nm suggests binding of deoxycholate to kallikrein at or around the tyrosine residue(s) or changes in the microenvironment of these residue(s).     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Reconstitution of fusogenic Sendai virus envelopes by the use of the detergent chaps

Sendai virus envelopes have been a useful tool in studying the mechanism of membrane-membrane fusion and have served as a vehicle for introducing foreign molecules (e.g., membrane proteins) into recipient cells. Reconstituted Sendai virus envelopes are routinely obtained following solubilization of virus particles with Triton X-100. This detergent has a low critical micellar concentration which precludes it from being the best detergent of choice in reconstitution studies. Nevertheless, it has remained in use since other detergents such as sodium deoxycholate and sodium cholate rendered the resultant vesicles inactive. Triton X-100 may be suboptimal for studies of some proteins that need be coreconstituted with the viral envelopes. Thus, alternative advantageous detergents, which retain the envelope fusogenic activity, have been sought. In this study we show that the synthetic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) effectively solubilizes the Sendai virions, and that the vesicles formed by simple reconstitution protocols appear structurally and biochemically similar to those obtained with Triton X-100. The resultant vesicles retain functional integrity as assessed in both fusion and hemolysis assays. This protocol seems to be useful in sendai envelope-mediated reimplantation of Fc epsilon receptors into the plasma membranes of rat basophilic leukemia cells.     

Inhibition of Aflatoxin Production by Surfactants

The effect of 12 surfactants on aflatoxin production, growth, and conidial germination by the fungus Aspergillus flavus is reported. Five nonionic surfactants, Triton X-100, Tergitol NP-7, Tergitol NP-10, polyoxyethylene (POE) 10 lauryl ether, and Latron AG-98, reduced aflatoxin production by 96 to 99% at 1% (wt/vol). Colony growth was restricted by the five nonionic surfactants at this concentration. Aflatoxin production was inhibited 31 to 53% by lower concentrations of Triton X-100 (0.001 to 0.0001%) at which colony growth was not affected. Triton X-301, a POE-derived anionic surfactant, had an effect on colony growth and aflatoxin production similar to that of the five POE-derived nonionic surfactants. Sodium dodecyl sulfate (SDS), an anionic surfactant, and dodecyltrimethylammonium bromide, a cationic surfactant, suppressed conidial germination at 1% (wt/vol). SDS had no effect on aflatoxin production or colony growth at 0.001%. The degree of aflatoxin inhibition by a surfactant appears to be a function of the length of the hydrophobic and hydrophilic chains of POE-derived surfactants.     

Effects of Four Detergents on the Oxidative and Phosphorylating Capacities of Potato Mitochondria

Oxidative and phosphorylating capacities of potato mitochondriawere studied with an oxygen electrode; succinate was used asa respiratory substrate. When low concentrations (0·1mg ml^–1 or less) of four detergents (Triton X-100, Tween80, sodium cholate, and sodium deoxycholate) were added intothe medium, the respiratory rates, respiratory controls, andADP/O ratios of mitochondria were increased. At higher concentrations,Triton X-100 and sodium deoxycholate were sharp inhibitors ofthe mitochondrial respiration; sodium cholate and over all Tween80 were much less toxic for potato mitochondria.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Erythrocyte hemolysis by detergents--a mathematical model and analysis of the concentration and kinetic curves

A mathematical model of erythrocyte lysis by detergents is developed which takes into consideration the kinetics of detergent binding to plasma membrane. Experimentally obtained sigmoidal kinetic and concentration curves of hemolysis are well described by the model. A comparative study is carried out in terms of the model of hemolytic action for five detergents: Triton X-100, sodium dodecylsulfate, sodium deoxycholate, cetyltrimethylammonium bromide, and cetylpyridinium chloride. The amount of detergent which should be bound to an erythrocyte membrane to induce lysis was found to be roughly the same for all detergents studied. However, detergents vary in their affinity to the membrane. Cetylpyridinium displays the highest affinity (and consequently the highest hemolytic activity), whereas deoxycholate has the least one.     

Binding, activation, and solubilization of the Ca2+-ATPase from sarcoplasmic reticulum by nonionic detergents

Interactions between delipidated Ca2+-ATPase from sarcoplasmic reticulum and four nonionic detergents--dodecyl octaoxyethyleneglycol monoether (C12E8), Triton X-100, Brij 58, and Brij 35--were characterized with respect to activation of ATPase activity, binding, and solubilization. C12E8 and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C12E8. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C12E8 and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C12E8 are more useful when bulk solubilization is the goal.     

Modulation of amino acid transport in preimplantation mouse embryos by low concentrations of non-ionic and zwitterionic detergents

In 4-cell embryos (but not in blastocysts), Triton X-100, a non-ionic detergent, stimulated leucine, phenylalanine, methionine and glutamic acid transport from 1.6 to 3.2-fold. All of these amino acids were transported exclusively by a sodium-independent mechanism. Triton X-100, however, did not stimulate the transport of other amino acids tested in 4-cell embryos. Furthermore, phenylalanine transport rates were stimulated about 2-fold at the 4-cell stage by all of the non-ionic and zwitterionic detergents tested at concentrations which were approximately one-tenth of the critical micellar concentration for each detergent. These concentrations did not block development, disrupt the cells, or make the cell membranes freely permeable. At the blastocyst stage, Z312, a zwitterionic detergent, inhibited the transport of phenylalanine and alanine and stimulated the transport of lysine, a pattern previously found to be linked to the sodium-dependent amino acid transport mechanism. We suggest that Z312 may be acting upon some component of sodium-dependent amino acid transport in blastocysts. The non-ionic and zwitterionic detergents seemed to have a common effect on amino acid transport in 4-cell embryos but elicited varied transport responses from blastocysts. These differential responses to detergents by blastocysts may reflect intrinsic changes in membrane composition and/or organization which occur during the normal course of preimplantation development.     

Uncoupling of oxidative phosphorylation by fatty acids and detergents suppressed by ATP/ADP antiporter inhibitors

The effects of ATP/ADP-antiporter inhibitors on the uncoupling of oxidative phosphorylation by palmitic acid, detergents and protonophore FCCP in liver mitochondria were studied. The uncoupling activity of these compounds was estimated by their stimulating effect on succinate oxidation and H+ conductivity of the inner mitochondrial membrane in the presence of oligomycin. Carboxyatractylate and pyridoxal 5-phosphate suppressed the uncoupling effects of palmitic acid and anionic detergents but had no effect on the uncoupling action of the nonionic detergent Triton X-100, the cationic detergent CTAB and FCCP. The data obtained are discussed in terms of the putative role of the ATP/ADP-antiporter in the electrophoretic transport of hydrophobic anions from the mitochondria.     

Electrical properties and glucose permeability of bilayer lipid membranes on incorporation of erythrocyte membrane extracts

1. Extracts of the human erythrocyte membrane have been prepared by solubilization with Triton X-100 and analysed by electrophoresis and gel filtration techniques. 2. The extracts have been incorporated asymmetrically into lecithincholesterol-n-decane planar bilayers. 3. The electrical characteristics and glucose permeabilities of the bilayers have been measured. 4. The extracts increased the electrical conductance of the bilayers and also markedly enhanced the D-glucose permeability but not the L-glucose permeability. 5. The enhanced D-glucose permeability was inhibited by monosaccharide transport inhibitors. 6. The results support the claim that a monosachharide facititated diffusion system has been set-up in vitro which has many of the characteristics of the transport system in the human erythrocyte membrane. 7. The data indicates that the trans membrane polypeptides of band 3 of the electrophoretogram of the erythrocyte membrane proteins (notation of Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2616) are implicated in D-glucose transport, although the possibility that relatively minor component of the membrane could be responsible for glucose transport cannot be eliminated.     

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.