Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.     

The Growth of the Primary Roots and Root Hairs of Sinapis alba and Lepidium sativus in Triton X-100

Seeds of Lepidium and Sinapis were germinated and grown for 3 days in different concentrations of Triton X-100 (0.001–0.1 % v/v). The elongation of the primary root was slightly stimulated by low concentrations. In concentrations above 0.01 %, Triton inhibited root growth and forked root hairs developed. The hairs elongated both at the apex and at the base, exhibited protoplasmic streaming and activity of particulate non-specific esterase. In contrast the growth of the hypocotyl of both Lepidium and Sinapis diminished steadily in increasing concentrations of Triton. Triton also affected the percentage germination of Lepidium, which increased or decreased according to the concentration used. The changes in root growth and germination and the appearance of branched root hairs in abundance coincide with a change in the detergent solution from monomer to aggregated molecules.     

The effect of detergent Triton X=100 on the light induced changes in the fluorescence yield of chloroplasts]

It is shown that light induced changes of the fluorescence yield (delta F) of isolated chloroplasts are affected by Triton X-100. delta F value descreases with the increase of the detergent concentration from 0 to 0.03%, increases in the range of 0.03--0.05% and is irreversibly blocked at concentrations more than 0.08--0.1%. The same dependence of delta F on the detergent concentration is obtained for "digitonin" fragments of chloroplasts enriched in the photosystem 2, but not for fragments enriched in the photosystem 1. Light induced delta F of chloroplasts treated by detergent were activated by hydroxylamine and saturated at lower light intensities than delta F of untreated chloroplasts. Addition of 0.01% Triton resulted in an activation of light induced delta F of chloroplasts with damaged donor part of photosystem 2. It is suggested that the complex dependence of delta F of chloroplasts on the Triton concentration is due to superposition of several effects: the uncoupling of photophosphorylation, inactivation of the electron transport chain in the donor and acceptor parts of photosystem 2, and changes of acting concentration of Triton X-100 within the range of critical micelle concentration.     

The interaction of detergents with bilayer lipid membranes

Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025–0.15 mM; dodecyl sulphate 0.82 in the range 0.01–1 mM; sodium deoxycholate 1.03 in the range 0.01–1 mM and sodium cholate 1.37 in the range 0.1–10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.     

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.     

Solubilization and partial purification of an enzyme involved in rat liver microsomal fatty acid chain elongation: beta-hydroxyacyl-CoA dehydrase.

The solubilization and partial purification of beta-hydroxyacyl-CoA dehydrase from rat liver microsomes has been accomplished through deoxycholate solubilization, ammonium sulfate fractionation, and ion exchange chromatography. A purification of about 90-fold based on total soluble activity was achieved, with an overall yield of 40%. However, the initial solubilization is accompanied by the loss of the secondary portion of the v/s curve observed with intact microsomes. The enzyme requires detergent during the purification procedure to remain "soluble," and is strongly activated by the inclusion of Triton-X-100 at concentrations above its critical micelle concentration in the assay mixture. In addition a preference for micelles has been inferred based on discontinuities in the v/s curves relative to the measured critical micelle concentration of the substrates in the absence of Triton X-100. Kinetic parameters calculated on the basis of micelle-specific activity indicated that beta-hydroxyacyl-CoA substrates possessing even-numbered alkyl chains from 14 to 20 carbon atoms differed little in Vm', but had progressively larger Km' as the chain length increased. The partially purified preparation was also active with beta-hydroxy-8,11-eicosadienoyl-CoA; and with 2-trans-enoyl-CoA substrates in a reverse (hydration) reaction.     

Biphasic interaction of Triton detergents with the erythrocyte membrane.

Octylphenoxy polyoxyethylene ethers (Triton detergents) interact with the erythrocyte membrane in a biphasic manner, i.e. they stabilize erythrocytes against hypo-osmotic haemolysis at low concentrations (0.0001-0.01%, v/v), but become haemolytic at higher concentrations. This biphasic behaviour was demonstrated with Triton X-114, Triton X-100 and Triton X-102. However, a critical chain length is a prerequisite for the haemolytic effect, because Triton X-45, which differs from the other Tritons only by the shorter chain of the polyoxyethylene residue, does not exhibit this biphasic behaviour, but goes on protecting against osmotic rupture up to saturating concentrations. Even a 1% solution of Triton X-45 does not cause haemolysis. This structural specificity of Triton X-45, namely the lack of haemolysis and efficient stabilization against osmolysis even at higher concentrations of the detergent, is exhibited at 0 degree and 37 degrees C as well as at room temperature. Three conclusions are reached: (i) a critical chain length of the octylphenoxy polyoxyethylene ethers is required for the haemolytic effect; (ii) the different structural requirements would suggest that different mechanisms are responsible for the haemolytic and the stabilizing effect of amphiphilic substances; (iii) the results suggest that haemolysis is not caused simply by dissolution of the membrane by the detergent but is a rather more specific process.     

Triton X nonionic surfactants reversibly inhibit the EDTA/KC1 ATPase activity of myosin

The nonionic octylphenoxy polyethoxy series of surfactants, Triton X, reversibly inhibited the $\text{EDTA}\text{KCl}$ ATPase activity of purified rabbit skeletal muscle myosin at concentrations at or below their reported critical micelle concentrations. The maximum degree of enzyme inhibition increased with ethoxy content to 88% (with Triton X 102 average ethoxy content, 12.5 per molecule). The results suggest that binding of the surfactant to the myosin molecule occurs below the critical micelle concentrations and that the hydrophilic ethoxy chain forms a diffusion barrier against approach of ATP to the enzyme's active site. This model has implications for the organization of myosin in the plasma membrane of phagocytes.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.     

Molecular organization of pheophytin a in aqueous solutions of detergents

Absorption, fluorescence and excitation fluorescence spectra of pheophytin a have been measured in aqueous solutions of nonionic (Triton X-100), anionic (sodium lauryl sulphate) and cationic (Cetyl pyridinium chloride) detergents at different concentrations and pH after system relaxation. By measuring the second derivative and differential spectra, it has been shown, that if detergent concentrations are lower than critical micelles concentration, or if the detergent is completely absent, the pigment forms conglomerates containing both dimeric and monomeric forms with an efficient energy transfer between them. If detergent concentrations are higher than critical micelles concentration, pheophytin a localizes in detergent micelle in monomeric form at neutral and acidic pH, and allomerizes at alkaline pH. The spectral characteristics of pheophytin a dimers in the conglomerate and its monomers in micelles poorly (if at all) depend on the sign of the detergent molecule charge.     

Phase separation of Triton X-100 micelle solution induced by osmotic stress

We have found out that the phase separation of Triton X-100 micelle solution was caused by the addition of poly(ethylene glycol) (PEG) above a critical concentration. The critical concentration of PEG depended on its molecular weight and temperature, larger molecular weight or higher temperature giving lower critical concentration. These results were analyzed on the basis of the osmoelastic coupling theory recently proposed by us (Biochemistry (1989) 28, 3710-3715; Biochemistry (1989) 28, 5626-5630).     

Effect of surfactant on the estimation by solid phase microextraction of bioavailable pyrene in soil samples

This study was focused on the effect of the presence of surfactant on the bioremediation efficacy and sensitivity of solid phase microextraction (SPME) in the pyrene-contaminated soil. Soils with 1.3 and 7.6% soil organic matter (SOM) were tested for biodegradation by microorganisms and extracted by aqueous solutions of the matrix used for SPME. For the biodegradation test, the presence of Triton X-100 at 5× CMC (critical micelle concentration) significantly enhanced pyrene removal for soil with lower SOM content (1.3%). However, this removal was insignificant for soil with higher SOM content (7.6%). The results may suggest that 5× CMC was not sufficient to improve significantly pyrene desorption for soil with higher SOM content. For the bioavailability test, in the absence of Triton X-100, SPME estimation of bioavailability in soils with indigenous or seeded microorganisms had an error range within 15%. However, with addition of Triton X-100, SPME estimations showed a significant decline (41 and 77%), in relation to their predicted values, for soil samples with SOM of 1.3 and 7.6%, respectively. The main reason for this underestimation is that micelle formation from the application of surfactant impacted the concentration of dissolved pyrene, rather than competitive site occupation between pyrene and surfactant molecules for SPME fiber. Thus, if soil samples contain surfactant, SPME would significantly underestimate bioavailability and risk level of PAH-contaminated sites.     

The effect of detergents on bovine cytochrome c oxidase: a kinetic approach

(1) Investigation of the relationship between the detergent concentration and steady-state and pre-steady-state kinetics of cytochrome c oxidase proved to be a valid approach in the study of protein-detergent interaction. (2) Laurylmaltoside, sodium cholate and Triton X-100 influenced the kinetics of cytochrome c oxidase cooperatively at detergent concentrations near their critical micelle concentration. This mode of interaction reflects disaggregation of the oxidase as a result of cooperative binding of the detergent. (3) Addition of increasing concentrations of Tween-80 to the aggregated enzyme caused a more gradual decrease in aggregation of the oxidase, which did not result in a change in activity of the enzyme. This suggests that aggregation of cytochrome c oxidase occurs in a highly regular manner in which no catalytic sites are shielded off. (4) Oxidase aggregates present at detergent concentrations below the critical micelle concentration of laurylmaltoside and Triton X-100 showed considerable activity. Their kinetics were equal to those of the oxidase in Tween-80, suggesting that the protein molecules are aligned in a similar way in all oligomers. Aggregates present in low concentrations of sodium cholate showed turnover rates that were twice as low as those observed with other aggregates. (5) Solubilisation of the oxidase by sodium cholate or Triton X-100 resulted in almost complete inhibition of enzymic activity, whereas the association rate of ferrocytochrome c was almost equal to that found for monomeric oxidase in laurylmaltoside. These results are in agreement with a mixed-type inhibition.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Effect of ethanol and methanol on the motility of Saccostrea commercialis sperm and sperm models

The organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found to significantly (P < 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X-100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and cAMP. Concentrations of 0.1% Triton X-100 demembranated sperm while 0.01% and 0.05% Triton X-100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 mM ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P < 0.001) decrease track velocity of 0.1% Triton X-100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tailwave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X-100 permeabilized sperm models. Reactivated motility of 0.05% Triton X-100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used.     

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.     

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity.

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

Quantitative determination by derivative spectrophotometry of Triton X-100 in solubilized preparations of membrane proteins

The amount of the nonionic detergent Triton X-100 in protein preparations cannot be determined spectrophotometrically from the normal spectrum because the spectral bands of the detergent and proteins overlap, but it was found that the detergent could be determined accurately by 4th-derivative spectrophotometry. The intensity of the derivative absorbance difference between the positive and negative peaks at about 277.5 and 281 nm, respectively, increased linearly with increase in the detergent concentration at more than its critical micelle concentration (about 0.03%). Proteins had little effect on these spectral bands of the detergent. This method is very simple and accurate for determination of the concentration of Triton X-100 in solubilized preparations of membrane proteins.     

Effects of ionic and nonionic detergents on antigen-antibody reactions.

Using highly sensitive and quantitative radioimmunoassay procedures we have measured the effects of different concentrations of three commonly used detergents, SDS, DOC, and Triton X-100, on antibody-antigen reactions. Triton X-100, had a relatively mild effect on primary antigen-antibody bindings, the precipitin reaction, and a double antibody RIA as evidenced by only an 8 to 10% inhibition of binding or precipitation. These results were not detergent concentration dependent, as Triton concentrations ranging from 5 to 0.1% had virtually no differential effects. Sodium deoxycholate (DOC) had a more profound effect on both primary antigen-antibody binding and the precipitin reaction than did Triton X-100, and its effects, unlike those of Triton X-100, were concentration dependent. There was a direct relationship between concentration of DOC and degree of inhibition of both primary binding and immune precepitation especially in antigen excess. Sodium dodecylsulfate (SDS), at concentrations 10- to 100-fold less than either Triton X-100 or DOC, had profound inhibitory effects on primary antigen-antibody binding, the precipitin reaction, and a double antibody radioimmunoassay. Generally, at concentrations greater that 0.01% SDS, almost all immunochemical reactivity is destroyed.