Studies on Triton X-100 detergent micelles.

Triton X-100 micelle formation at 25 degrees C was studied by use of sedimentation equilibrium and fluorescence spectroscopic techniques. The apparent molecular weight of the major Triton X-100 micelle was found to be 81250, indicating a micelle number of 125. A micelle number of 121 was obtained with fluorescence titration experiments, which showed one molecule of 1-anilino-8-naphthalene sulfonate binding per micelle with an apparent association constant of 0.9 x 10(5) M. The fluorescent titration experiments also indicated the presence of another TX-100 binding species of variable size.     

The interaction of phosphatidylcholine bilayers with Triton X-100

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.     

Spectroscopic properties of protochlorophyll forms in Triton X-100 detergent micelles

Protochlorophyll (PChl) forms were performed in Triton X-100 detergent micelles. The concentration of Triton X-100 was 7·10^?4 M (above the critical micellar concentration); the concentration of PChl varied between 1.6·10^?5 and 1.8·10^?4 M. Absorption, fluorescence and circular dichroism (CD) spectra were registered. The absorption spectra were resolved into Gaussian components by computer analysis. PChl forms with absorption bands at 632–634, 638, 652–654, 663–664, 668 and 676 nm and with fluorescence emission bands at 634–636, 640–644, 652–655, 677–678, 686 and 694–696 nm were observed in micellar solutions of different PChl concentrations. The CD spectra showed a strong dependence on the concentration of PChl: positive CD signals or positive Cotton effects were observed in the vicinity of 650 nm. The intensity of these signals increased in parallel with increasing concentration of PChl. No CD signals were found in the region of the longer wavelength absorption bands. These data show that the PChl exists in many different forms in this system, and the spectroscopic properties of these forms are determined by different molecular interactions viz., interactions of PChl with Triton X-100 or water molecules and/or by the aggregation of PChl.     

Structural changes induced by Triton X-100 on sonicated phosphatidylcholine liposomes

Solubilization of sonicated unilamellar vesicles by Triton X-100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid:detergent mole ratio, about one-third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two-thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes.     

Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100

Sonicated unilamellar and large multilamellar liposome suspensions have been treated with the non-ionic detergent Triton X-100, and the subsequent changes in turbidity have been studied as a function of time. Sonicated liposome suspensions exhibit an increase in turbidity that takes place in two stages, a fast, low-amplitude one is completed in less than 100 ms, and a slow large-amplitude one occurs in 20-40 s. The first increase in turbidity is associated to detergent incorporation into the bilayer, and the second one, to vesicle fusion. The fast stage may be detected at all detergent concentrations, while the slow one is only seen above the critical micellar concentration of Triton X-100. Both processes may be interpreted in terms of first-order kinetics. Studies of the variation of kexp with lipid and detergent concentration suggest a complex multi-step mechanism. In the case of multilamellar liposomes, a fast increase in turbidity is also seen after detergent addition, which is followed by a slow (20-60 s) decrease in turbidity and a very slow (up to 12 h) large scale decrease in turbidity. These processes do not conform to single-exponential patterns. The fast stage is also thought to reflect surfactant incorporation, while the decrease in turbidity is interpreted as bilayer solubilization starting with the outer bilayer (slow stage) and proceeding through the remaining ones (very slow stage).     

Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus.     

Interaction of beef liver lipase with mixed micelles of tripalmitin and Triton X-100

When concentrated dispersions of tripalmitin in Triton X-100 are added to reaction mixtures containing soluble beef liver lipase, the rate of hydrolysis of tripalmitin increases with incubation time. When the diluted substrate is aged at 37 degrees C for 3 hr before the addition of enzyme, the rate of hydrolysis is greater than the rate with freshly diluted dispersions and is constant for at least 2 hr. The reciprocal of the rate of hydrolysis is a complex function of the reciprocal of the substrate concentration when measured with freshly diluted substrate dispersions. A linear relationship between these reciprocals is obtained when measured with aged preparations of substrate. The rate and extent of increase of the velocity of hydrolysis of the aged substrate in relation to the velocity of hydrolysis of freshly diluted substrate are directly proportional to the substrate concentration and inversely proportional to the Triton X-100 concentration. The apparent V(max) of beef liver lipase for tripalmitin in diluted and aged dispersions is independent of the Triton X-100 concentration, while the apparent K(m) is inversely proportional to the Triton X-100 concentration. The apparent K(m) for tripalmitin complexes at zero Triton X-100 concentration was judged to be 7.5 x 10(-5) m. The molecular size of dispersion complexes does not change significantly as dispersions are aged. The spherical diameter of the complexes assessed by gel filtration techniques is in the order of 100 A.     

PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.     

Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidylserine

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids and diacylglycerols. Triton X-100 mixed micelles containing 8 mol % phosphatidylserine (PS) and 2.5 mol % sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1 did in the standard assay. However, protein kinase C activity was more responsive to diC18:1 in the mixed micellar assay than the standard assay. At 8 mol % PS and 100 microM Ca2+, diC18:1 stimulated maximally at 1 mol %. At 2.5 mol % diC18:1 and 100 microM Ca2+, PS did not activate until 3 mol % and then did so cooperatively with maximal stimulation occurring at 6-8 mol %. Direct evidence for a Ca2+-, PS-, and diC18:1-dependent interaction of protein kinase C with mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data permit inferences pertaining to the number of diC18:1 and PS molecules/micelle which are required for activation. For diC18:1, a single molecule may be sufficient but no more than 2 molecules are required. For PS, greater than 4 but less than 10 molecules are required. These data establish that a phospholipid bilayer is not required for protein kinase C activation and that activation of monomeric protein kinase C occurs.     

Kinetic analysis of yeast phosphatidate phosphatase toward Triton X-100/phosphatidate mixed micelles

A detailed kinetic analysis of purified yeast membrane-associated phosphatidate phosphatase was performed using Triton X-100/phosphatidate mixed micelles. Enzyme activity was dependent on the bulk and surface concentrations of phosphatidate. These results were consistent with the "surface dilution" kinetic scheme (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) where phosphatidate phosphatase binds to the mixed micelle surface before binding to its substrate and catalysis occurs. Phosphatidate phosphatase was shown to physically associate with Triton X-100 micelles in the absence of phosphatidate, however, the enzyme was more tightly associated with micelles when its substrate was present. The enzyme had 5- to 6-fold greater affinity (reflected in the dissociation constant nKsA/chi) for Triton X-100 micelles containing dioleoyl-phosphatidate and dipalmitoyl-phosphatidate when compared to micelles containing dicaproyl-phosphatidate. The Vmax for dioleoyl-phosphatidate was 3.8-fold higher than the Vmax for dipalmitoyl-phosphatidate, whereas the interfacial Michaelis constant chi KmB for dipalmitoyl-phosphatidate was 3-fold lower than the chi KmB for dioleoyl-phosphatidate. The specificity constants (Vmax/chi KmB) of both substrates were similar which indicated that dioleoyl-phosphatidate and dipalmitoyl-phosphatidate were equally good substrates. Based on catalytic constants (Vmax and chi KmB), dicaproyl-phosphatidate was the best substrate with an 11- and 14-fold greater specificity constant when compared to dioleoyl-phosphatidate and dipalmitoyl-phosphatidate, respectively.     

Cholesterol reverts Triton X-100 preferential solubilization of sphingomyelin over phosphatidylcholine: A P-NMR study

The distribution of phosphatidylcholine (PC) and sphingomyelin (SM) between the solubilized (micellar) and non-solubilized (lamellar) fractions arising from bilayers composed of PC and SM, with or without cholesterol (Chol) has been measured under conditions of partial, incomplete solubilization by Triton X-100. Quantitation is achieved by ³¹P-NMR determination of the composition of mixed micelles in the range of bilayer-micelle coexistence. We find that the solubilized fraction of bilayers consisting of binary mixtures of PC and SM is rich in SM, as expected from previous data on solubilization of pure PC and pure SM liposomes. In contrast, after partial solubilization of ternary mixtures of PC, SM and Chol, the solubilized fraction becomes SM-poor, as observed in the partial solubilization of biomembranes.     

Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X-100.

Effective detergent:lipid ratios (i.e. molar ratios in the mixed aggregates, vesicles or micelles) have been estimated for the solubilization of phosphatidylcholine vesicles by Triton X-100. Effective molar ratios are given for both the onset and the completion of bilayer solubilization; small unilamellar, large unilamellar and multilamellar vesicles have been used. Effective detergent:lipid ratios are independent of phospholipid concentration, and their use allows a deeper understanding of membrane-surfactant interactions.     

Adenine nucleotide translocase greatly increases the partition of trinitrophenyl-ATP into reduced Triton X-100 micelles.

The presence of adenine nucleotide translocase (ANT) was found to greatly enhance the partitioning of the ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) into reduced Triton X-100 micelles. The protein's effect was studied through the quenching of fluorescence of purified ANT, irreversibly inhibited by carboxyatractyloside (CAT), solubilized in reduced Triton X-100 micelles. The dependence of quenching of the protein's time-resolved tryptophan fluorescence on TNP-ATP concentration was measured and found to follow a Stern-Volmer mechanism. However, the calculated quenching constant was too large to be accounted for by the aqueous TNP-ATP concentration. Experiments were therefore conducted to determine the partitioning of the quencher between the three phases present: aqueous, protein-free micelle, and protein micelle; a system also described by the equation of Omann, G. M., and M. Glaser (1985. Biophys. J. 47:623-627.). By measuring the dependence of the apparent quenching rate constant on the protein concentration and protein/micelle ratios, this equation was used to calculate both the quencher partition coefficient into protein-free micelles (Pm) and into protein-micelles (Ppm), as well as the bimolecular quenching rate constant (kpm) in protein micelles. From the quenching experiments, kpm = 5.0 x 10(8)M-1s-1,Pm = 290 and pyrene quenching experiment to be 325, and by a rapid filtration experiment to be 450. Clearly, the presence of the integral membrane protein ANT-CAT in reduced Triton X-100 micelles greatly increases the partition of TNP-ATP into the micelle. ANT alters the properties and thus, the structure of the detergent micelle, which has direct implications for the use of detergent micelles as a model system for membrane proteins and may indicate that analogous effects occur in the mitochondrial membrane.     

The effect of cholesterol on the solubilization of phosphatidylcholine bilayers by the non-ionic surfactant Triton X-100

Most of the studies on the solubilization of model membranes by Triton X-100 (TR) involve one lipid. The aim of the present study was to evaluate the effect of the addition of cholesterol on the solubilization of bilayers made of palmitoyloleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). Detailed investigation of the kinetics of solubilization of the cholesterol-containing bilayers by TR at different temperatures reveals that: (i) At 4 degrees C, solubilization of both systems is relatively slow. Hence, in order to prevent misleading conclusions from turbidity measurements it is important to monitor the solubilization after steady-state values of optical density (OD) are reached. (ii) Studies of the temperature-induced changes of the aggregates present in mixtures of TR, POPC and cholesterol indicate that the state of aggregation at all temperatures (including 4 degrees C) represents equilibrium. By contrast, for DPPC/cholesterol/TR mixtures "kinetic traps" may occur not only at 4 degrees C but at higher temperatures as well (e.g. 37 degrees C). (iii) The presence of cholesterol in POPC bilayers makes the bilayers more resistant to solubilization at low temperatures, especially at 4 degrees C. As a consequence, the temperature dependence of the TR concentration required for complete solubilization (Dt(sol)) is no longer a monotonically increasing function (as for POPC bilayers) but a bell-shaped function, with a minimum at about 25 degrees C. Inclusion of cholesterol in DPPC bilayers makes the bilayers more resistant to solubilization at all temperatures except 4 degrees C. In this system, we observe a bell-shaped dependence of Dt(sol) on temperature, with a minimum at 37 degrees C. (iv) Both the rate of vesicle size growth and the rate of the solubilization of POPC vesicles are not affected by the inclusion of cholesterol in the bilayers. Similarly, cholesterol did not affect significantly the rate of size growth of DPPC bilayers at all temperatures, but reduced the rate of solubilization at 4 degrees C.     

Triton X-100 in Giemsa Staining of Blood Parasites

The effects of a surface active agent, Triton X-100, were studied in the routine Giemsa staining of seven blood parasites: Plasmodium vivax, Trypanosoma cruzi, T. lewisi, Leishmania donovani, Toxoplasma gondii, and microfilariae of Dirofilaria immitis and of Wuchereria bancrofti. Concentrations of Triton X-100 ranging from 0.01% to 0.5% were used in staining (a) both thick and thin blood films of all organisms except L. donovani, (b) tissue smears of L. donovani, and (c) tissue and peritoneal fluid smears of T. gondii. In general, the addition of the detergent to the Giemsa solution resulted in cleaner preparations and better stained organisms. Morphological details were more distinct, thus facilitating microscopical detection and identification of species. The most beneficial concentration of Triton X-100 was found to be 0.1%. Since it has a hemolytic effect on erythrocytes, concentrations above 0.01% cannot be used in staining thin blood films. It is suggested also that the use of Triton-Giemsa may help prevent transfer of some of these organisms from one slide to another during mass staining procedures as it has been demonstrated to do with malaria parasites (Brooke and Donaldson, 1950).     

Permeabilization of microorganisms by Triton X-100.

A simple permeabilization procedure has been developed which allows the reliable determination of enzyme activitiesin situ in a variety of different microorganisms. Permeabilization is obtained by freezing cell suspensions in the presence of a low concentration of the anionic detergent Triton X-100. After thawing, the cells can be used directly in the enzyme assays. The procedure has been optimized using the yeastSaccharomyces cerevisiae. Yeast cells are completely permeabilized by Triton X-100 concentrations of 0.05% (v/v), and permeabilization is independent of cell age and cell concentration. The treatment makes the cells freely diffusible for macromolecules up to molecular weights around 70,000. Cytoplasmic and mitochondrial amino acid biosynthetic enzymes as well as aminoacyl-tRNA synthetases could be readily measured in treated cells. The method has been successfully applied to the determination of enzyme activities in other fungi as well as in gram-positive and gramnegative bacteria.