Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport.

A carrier protein mediating alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE- or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TFo) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.     

Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

A membrane protein of relative molecular mass (Mr) 127,000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and beta-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of Mr 127,000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127,000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127,000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5/5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for beta-lactam antibiotics and small peptides of Mr 127,000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells.

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine prefferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

Surfactant Effects on the Permeability of Photosynthetic Membrane from Rhodobacter sphaeroides 2.4.1 Probed by Electrochromic Shift of Endogenous Carotenoids

Four surfactants, sodium cholate(SC), n-dodecyl-β-D-maltopyranoside(DDM), lauryldimethylamine oxide(LDAO) and Triton X-100(TX), which are generally used in photosynthetic pigment-protein complexes preparation, were studied on their interaction with photosynthetic membrane from Rhodobacter sphaeroides 2.4.1 by electrochromic absorption band-shift of endogenous carotenoids and by vesicle size measurements as well. The surfactant critical micelle concentration(cmc) was found to be negatively correlated with the capability of enhancing the permeability of photosynthetic membranes to proton, and more elaborated model of surfactants interacting with membranes was obtained. The electrochromic absorption band-shift measurement might develop into a useful tool to evaluate the effects of surfactants on various membranes.     

The HLB dependency for detergent solubilization of hormonally sensitive adenylate cyclase.

The HLB dependency for the solubilization of membrane proteins and adenylate cyclase activity from a plasma membrane-enriched fraction from rat liver has been determined. The HLB (hydrophilic/lipophilic/balance) number of a detergent is an empirical measure of its relative hydrophobicity. Detergent HLB numbers vary systematically with the length of the ethylene oxide chain for a homologous series of detergents such as the Triton X series. These detergents have a constant hydrophobic moiety, octylphenyl, and a variable polar portion, polyethoxyethanol. Basal-NaF-epinephrine-, and glucagon-stimulated adenylate cyclase activities were solubilized in the HLB range of 16.8-17.4. Solubilization was most effective in 0.01 M Tris buffers at pH 7.5 containing 1-5 mM mercaptoethanol, 1 mM MgCl2, and 0.1% Triton X-305. The detergent to membrane protein ratio used in these studies was 3:1. Criteria for solubilization included lack of sedimentation at 100,000 X g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cyclase activity in Sephadex G-200 gels. The apparent molecular weight of the solubilized enzyme was approximately 200,000 in the presence of Triton X-305. The solubilized enzyme was stimulated 5-fold by NaF, 7-fold by glucagon, and 20-fold by epinephrine compared to the particulate enzyme used in this study which was stimulated 10-fold, 3.4-fold, and 4-fold by NaF, epinephrine, and glucagon, respectively. The solubilized enzyme is stable for several weeks when stored at -60 degrees C.     

Solubilization and purification of galactosyltransferase from golgi membranes of rat ventral prostate

UDP-galactose ovomucoid galactosyltransferase, a membrane-bound enzyme involved in the biosynthetic pathways for formation of the nonreducing terminal oligosaccharide sequences on glycoproteins, has been solubilized and purified from rat ventral prostate Golgi membranes. Solubilization was effected by treatment of the particulate fraction with Triton X-100 (0.5% v/v) and MnCl₂ (25 mM). The solubilized enzyme was purified by affinity chromatography on hen ovomucoid-sepharose column. The purified galactosyltransferase showed three protein bands of approx. 74,000, 60,000, and 54,000 daltons on sodium dodecyl sulfate gel electrophoresis. On gel filtration, enzyme activity eluted at approx. 70,000 daltons with a broad shoulder between 60,000 and 50,000 daltons. Isoelectric focusing of the purified enzyme resolved at least five active bands with pHi of 9, 7.4, 6.75, 6.1, and 4.8.     

AOT/Triton X-100混合反胶束体系中假丝酵母脂肪酶催化蓖麻油水解的活性

研究了二(2-乙基己基)琥珀酸磺酸钠(AOT)/Triton X-100混合反胶束体系中假丝酵母脂肪酶(candida rugosa lipase)催化蓖麻油水解的反应. 考察了Triton X-100占总表面活性剂的摩尔分数(x(Triton X-100))、水与总体表面活性剂的摩尔比(ω0)、pH值、反应温度以及底物蓖麻油的浓度等因素对酶活性的影响. 研究结果表明, 加入非离子表面活性剂Triton X-100可以使假丝酵母脂肪酶的活性得到显著提高, 但是当底物蓖麻油的浓度大于0.24 mol·L-1时, 会对假丝酵母脂肪酶产生抑制作用.     

Effects of micelle properties on the conformation of oligocholates and importance of rigidity of foldamers

The conformation of a cholate hexamer with a clicked tether in between two tricholate units and pyrene groups at the chain ends was studied by fluorescence spectroscopy. In contrast to the parent cholate hexamer that folded in all micelles investigated, the folding of the clicked hexamer was highly dependent on the type of surfactant used to solubilize the compound. The clicked oligocholate folded in the Brij 35 micelle, possibly due to the latter's small size and strong internal hydrophobicity. The oligocholate formed intermolecular aggregates in SDS solutions below the CMC of the surfactant. The aggregates were dissociated by the SDS micelles but the individual oligocholate stayed unfolded. In Triton X-100 and sodium cholate solutions, the aggregated, unfolded, and folded oligocholates coexisted and gradual unfolding occurred with an increasing concentration of the surfactant. The conformation of the clicked oligocholate was sensitive to the nonideal mixing of ionic/nonionic micelles and to the unconventional aggregation of sodium cholate.     

Action of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis is significantly influenced by coexisting lipids in substrate-detergent micelles.

The effects of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol on the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail in phosphatidylinositol (PI)/detergent mixed micelles. By addition of PC, the enzymatic hydrolysis of PI was significantly stimulated in PI/Triton X-100 as well as PI/sodium deoxycholate (SDC) mixed micelles. SM stimulated enzyme activity toward PI/Triton X-100 micelles at a lower molar ratio of SM to PI, but was rather inhibitory at a ratio higher than 2.0. The enzyme activity became significantly lower with an increase of PE or cholesterol in PI/Triton X-100 micelles. Actually, both PE and cholesterol were intensively inhibitory when added at a higher molar ratio to PI in Triton X-100-containing micelles. In the system of PI/SDC mixed micelles, not only PC but also SM, PE and cholesterol enhanced the enzymatic hydrolysis of PI. The difference between PI/Triton X-100 and PI/SDC micelles regarding the effects of these lipids on PI-PLC action, must be dependent on the physical state of micelles formed by these detergents and lipids.     

丁二酸二异辛酯磺酸钠(AOT)/TritonX-100混合反胶束体系中纤维素酶降解纤维素的研究

对于底物不溶于水的纤维素降解反应而言,为了增强纤维素酶的活性,在丁二酸二异辛酯磺酸钠(AOT)/异辛烷反胶束体系中加入非离子表面活性剂TritonX-100进行纤维素降解实验.结果表明,在AOT中加入非离子表面活性剂TritonX-100可以使纤维素酶的活性提高,非离子表面活性剂TritonX-100与AOT的最佳物质的量之比是0.20.考察了水与表面活性剂的物质的量之比(Wo)、不同酸度(pH)和不同温度(T)等其他反应条件对纤维素降解反应的影响.研究结果表明,反应的最佳条件是:Wo为3.3,T为315.11K,pH为5.10.     

Optical properties of Triton X-100-treated purple membranes embedded in gelatin films

The purple membrane (PM) of the microorganism Halobacterium salinarium contains a hexagonally packed monolayer of the light-sensitive protein, bacteriorhodopsin (BR). The optical characteristics of gelatin-immobilized PMs depend strongly on the chemical environment of the PMs in the matrix. Here we present photoinduced absorptive and holographic characteristics of gelatin-embedded PMs solubilized with the non-ionic detergent, Triton X-100. The BR/detergent interaction was shown to slow the M-to-initial state transition of the photocycle and to increase the photosensitivity of the BR films. The lifetime of the holographic grating in Triton X-100-treated BR films was 2–3 times greater, when compared to the unmodified sample. Holographic grating growth times in BR films were shown to change depending on the extent of solubilization. The measured holographic sensitivity appeared to maximize in the range of Triton X-100/BR molar ratios from 15:1 to 25:1. The possible advantages of solubilized PM films as they are applied to optoelectronic devices are discussed.     

Solubilization of cellular membrane proteins from Streptococcus mutans for two-dimensional gel electrophoresis

Membrane proteins are rarely identified in two-dimensional electrophoretic (2-DE) proteomics maps. This is due to low abundancy, poor solubility, and inherent hydrophobicity leading to self-aggregation during the first dimension. In this study, membrane proteins from the Gram-positive bacterium Streptococcus mutans were solubilized using three different methods and evaluated by 2-DE. In the first method, the extraction was performed using sodium dodecyl sulfate (SDS) followed by solubilization with a chaotropic buffer and precipitation with methanol/chloroform. The second method was based on temperature-dependent phase partitioning using Triton X-114 followed by purification using the ReadyPrep 2-D clean-up kit from Bio-Rad. The third method involved extraction using the organic solvents trifluoroethanol (TFE) and chloroform, which produced three separate phases. The upper aqueous phase, enriched with TFE, gave the highest overall protein yield and best 2-DE resolution. Protein spot identification by nanoelectrospray quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS) revealed known membrane and surface-associated proteins. This is the first report describing the successful solubilization and 2-D electrophoresis of membrane proteins from a Gram-positive bacterium.     

Alterations in the Detergent-Induced Membrane Permeability and Solubilization of Saturated Phosphatidylcholine/Cholesterol Liposomes: Effects of Poly(ethylene glycol)-Conjugated Lipid

We have investigated the effects of two bile salts, chenodeoxycholate (CDC) and ursodeoxycholate (UDC), and a widely used detergent, Triton X-100 (T(X-100)), on normal and poly(ethylene glycol)-modified liposomes (PEGylated liposomes). We tested various lipid compositions, including hydrogenated soybean phosphatidylcholine/cholesterol/PEG-conjugated lipid (HSPC/PEG-lipid). Alterations in permeability were determined by the rate of drug release from the liposomes and solubilization was assessed by measuring the particle size of liposomes. In addition, we attempted to observe interactions between the detergents and lipid bilayers by using surface plasmon resonance (SPR). CDC induced drug release from liposomes in a dose-dependent manner, and the PEGylated liposomes tended to be susceptible to CDC. While UDC did not strongly induce drug release from liposomes, UDC exhibited a similar tendency with CDC. In case of T(X-100), there were significant differences in the percentage of released drug between normal and PEGylated liposomes, and the percentage of T(X-100)-induced drug release further increased with an increased ratio of PEG-lipid. SPR analysis revealed that the lipid bilayer including PEG-lipid was selectively solubilized by T(X-100), correlating with the drug release data. These results suggest that the effect of detergents on the lipid bilayer of liposomes depends on both the kind of detergent and the lipid composition, including the presence or absence of PEG-lipid. Moreover, the effects of T(X-100) on the lipid bilayers of the PEGylated liposomes significantly differed from those on the lipid bilayers of the normal liposomes.     

PHOTOREACTIVITY OF ISOLATED PHOTOSYSTEM I PARTICLES UPON COMBINATION WITH ARTIFICIAL LIPID MEMBRANES OR TRITON X-100 MICELLES*

Abstract— PS-I particles isolated according to Shiozawa et al. (1974) show increased rates of O₂-_- and H ⁺-uptake with ascorbate as electron donor upon combination with an artificial vesicular lipid membrane. The amount of increase varies depending on the reconstitution procedure used. Combination of PS-I particles with Triton X-100 micelles increases these photochemical activities even more. The observed proton uptake in PS-I lipid vesicles is not caused by the well-known proton gradient found in thylakoid membranes, since lipid vesicles containing extracted leaf Chl show the same activities and uncouplers have no effect. Because these phenomena are also caused by solubilized Chl, it is concluded that there is no obvious correlation with PS-I activity. Proton uptake most probably is caused by oxidation of ascorbate by either singlet oxygen, superoxide or OH-radicals formed in the light. Experimental results are obtained which indicate that Chl in lipid catalyzes formation of superoxide and singlet oxygen. However, it is not clear whether superoxide formation is caused by direct electron transport from excited Chl to oxygen or by a secondary reaction. Diphenylcarbazone disproportionation has been reported as a specific photosystem I reaction. However, PS-I lipid vesicles and Chl-lipid-Triton X-100 mixtures oxidize DPCN at comparable rates, showing that the reaction is not specific for PS-I. Cations stimulate DPCN disproportionation in Chl-lipid-Triton X-100 mixtures but do not affect the rate of P700 photooxidation at all. Therefore it is suggested that Gross and Greniers (1978) conclusion that cation regulation of PSI electron flow (studied by DPCN disproportionation of PS-I particles in Triton X-100 micelles) provides a fine tuning mechanism for energy transfer, has to be reevaluated.     

Membrane proteome analysis of the green-sulfur bacterium Chlorobium tepidum

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including membrane proteins, which are rarely detectable on two-dimensional electrophoresis gels. In this study, different methods were employed for the enrichment of membrane proteins from Chlorobium tepidum prior to analysis with two-dimensional electrophoresis (2-DE). Isolated membranes were solubilized with Triton X-100 and from the supernatant we identified 58 unique proteins. The use of ionic sodium dodecyl sulfate (SDS) for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of the identified proteins was increased to 117. The use of acetone for protein precipitation improved the results by extracting compounds potentially deleterious to the resolution of 2-DE. However, the additional proteins detected by the use of SDS are in the majority more difficult to solubilize than less hydrophobic proteins. Further our attempts for selective extraction of the outer membrane proteins using the acid glycine method allowed the identification of 37 proteins of which 14 were predicted to have a signal sequence indicating their localization in the periplasmic space or in the outer membrane.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium.

The alkaline phosphatase present on isolated brush border and basal lateral membranes of rat duodenal epithelium were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6--9.8, similar substrate km's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin of papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000--150,000, and both enzymes show an Rf value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The Vmax of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 mumoles/mg-h as opposed to 14 mumoles/mg-h. The differences in Vmax are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

A BLUE LIGHT-SENSITIVE CYTOCHROME-FLAVIN COMPLEX FROM CORN COLEOPTILES. FURTHER CHARACTERIZATION*

Abstract— The partially purified blue light-sensitive membrane-associated flavin-cytochrome complex from etiolated corn coleoptiles shows a unique sharp α-band at 555 nm in its light-minus-dark difference spectrum at liquid nitrogen temperature. This band is clearly distinguishable from the α-bands found in fractions enriched for mitochondria and endoplasmic reticulum respectively. The photoactive membrane fraction is shown to have ATPase activity that is not stimulated by K⁺ and that is not inhibited by oligomycin. Other than flavin fluorescence at 525 nm obtained upon excitation at 450 nm, there is a second fluorescent component with emission at 430 nm on excitation at 350 nm. The mid-point potential of the Triton X-100 solubilized b-cytochrome, measured by simultaneously monitoring the reduction of the pyocyanine 600-800 nm peak and the appearance of the 427 nm Soret peak of the b-cytochrome upon titration with dithionite in the presence of ferricyanide, is estimated to be ?65 mV. The kinetics of the blue light-induced reduction and dark rcoxidation of the 6-cytochrome suggest that the mid-point potential of the b-cytochrome is not affected by Triton X-100 solubilization.     

Physicochemical Investigations on the Interaction of Surfactants and Salts with Polyvinylpyrrolidone in Aqueous Medium

Microcalorimetric investigations have been carried out onthe interaction of the surfactants sodium cholate, sodium deoxycholate, tetradecyltrimethylammonium bromide, cetyl(hexadecyl)trimethylammonium bromide, and p-tert-octylphenoxy polyoxy-ethylene ether (Triton X-100) and the salts potassium iodide, sodium benzoate, sodium bromide, and sodium salicylate with the neutral polymer polyvinylpyrrolidone (PVP). The enthalpy of dilution of the surfactants has been measured in the absence and presence of the polymer and the results are compared to determine the effect of PVP on the micellization of the surfactants and the energetics of the process. As well, the micellization activity of the surfactants in the presence of the polymer has been studied by conductometric and fluorimetric methods. The enthalpy of dilution of the salts has been measured to provide an understanding of the nature and magnitude of their interaction with PVP. Copyright 2001 Academic Press.     

Purification of the membrane-form variant surface glycoprotein of Trypanosoma brucei

The membrane-form variant surface glycoprotein (mfVSG) is anchored in the plasma membrane of African trypanosomes by a diacylglycerol residue. On cell rupture the anchor is rapidly cleaved by an endogenous phospholipase C. A purification procedure is described which results in native mfVSG devoid of lipase activity. A total membrane fraction is prepared in the presence of the SH-inhibitor p-chloromercuribenzenesulphonic acid (pCMBS). Membrane proteins are solubilized in the presence of pCMBS and the detergent Zwittergent 3-12, conditions which inhibit the activity of the phospholipase. mfVSG is then purified by successive chromatography on rabbit anti-VSG affinity and cation-exchange columns (25% yield). The isolated protein is electrophoretically pure and partitions into the detergent phase on Triton X-114 phase separation, proving that it retains the diacylglycerol anchor.     

Cloud point extraction equilibrium of lanthanum(III), europium(III) and lutetium(III) using di(2-ethylhexyl)phosphoric acid and Triton X-100

The cloud point extraction behaviors of lanthanoids(III) (Ln(III) = La(III), Eu(III) and Lu(III)) with and without di(2-ethylhexyl)phosphoric acid (HDEHP) using Triton X-100 were investigated. It was suggested that the extraction of Ln(III) into the surfactant-rich phase without added chelating agent was caused by the impurities contained in Triton X-100. The extraction percentage more than 91% for all Ln(III) metals was obtained using 3.0 × 10⁻⁵ mol dm⁻³ HDEHP and 2.0% (v/v) Triton X-100. From the equilibrium analysis, it was clarified that Ln(III) was extracted as Ln(DEHP)₃ into the surfactant-rich phase. The extraction constant of Ln(III) with HDEHP and 2.0% (v/v) Triton X-100 were also obtained.