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At least 19 recordsLinked to original sources
PubMed1996

Brominated detergents as tools to study protein-detergent interactions.

In order to study protein-detergent short-range interactions, we analyzed the quenching by brominated detergents of reticulum sarcoplasmic (SR) Ca(2+)-ATPase intrinsic fluorescence. For this purpose, 7,8-dibromododecyl beta-maltoside and 2-O-(10,11-dibromoundecanoyl)sucrose, brominated analogs of two non-ionic detergents, the frequently used dodecylmaltoside and the newly synthesized 2-O-lauroylsucrose respectively, were prepared. Rayleigh scattering measurements showed that the brominated detergents efficiently and rapidly solubilized SR vesicles like their non-brominated analogs although at slightly higher concentrations. Similarly, each analog had a slightly higher critical micellar concentration than its parent detergent. The partition coefficient K (expressed as the ratio of the molar fraction of detergent in the SR lipid phase to that in the aqueous phase, at pH 7.5 and 20 degrees C) was similar for brominated and non-brominated dodecyl maltoside (3.5-4 x 10(5)) and slightly lower for dibromoundecanoylsucrose (approximately 10(5)) than for lauroylsucrose (approximately 2 x 10(5)). At detergent concentrations too low to solubilize the membrane, the brominated detergents rapidly inserted (within seconds) into SR vesicles. In this concentration range, Ca(2+)-ATPase fluorescence quenching steadily increased with detergent concentration. When the membrane was saturated with detergent, the residual fluorescence was about half of its initial value, indicating significant protein-detergent, contacts, possibly due to a slightly higher affinity of Ca(2+)-ATPase for these detergents than for phospholipids. For higher detergent concentrations, solubilizing the membrane, the fluorescence continued to decrease with detergent concentration, with no evidence for a dramatic change in the average hydrophobic environment of the protein during the transition from bilayers to a soluble state. For still higher detergent concentrations, above that necessary for membrane solubilization, the fluorescence was further quenched to a residual relative value of about 20%, corresponding to further delipidation of the protein surface, in agreement with previous results [de Foresta, B., le Maire, M., Orlowski, S., Champeil, P., Lund, S., Møller, J.V., Michelangeli, F. & Lee, A.G. (1989) Biochemistry 28, 2558-2567]. Fluorescence quenching for solubilized Ca(2+)-ATPase was quickly reversed upon addition of excess non-brominated detergent. The effects of the four detergents on the Ca(2+)-ATPase hydrolysis of p-nitrophenyl phosphate were similar and correlated with the protein-detergent contacts evidenced above. In conclusion, both these brominated detergents appear to be promising tools to study protein-detergent interactions at the hydrophobic surface of a membrane protein, either in a membrane or in solubilized complexes.

Animals↗
PubMed1984

Phenyl-Sepharose-mediated detergent-exchange chromatography: its application to exchange of detergents bound to membrane proteins.

Detergent-saturated phenyl-Sepharose was used to exchange detergents for one another in the presence of membrane proteins. The alkyl detergents lauryl maltoside, octyl glucoside, and dodecyl sulfate were each successfully exchanged for Triton X-100, Triton N-101, or Nonidet P-40 present in a solution of either cytochrome c oxidase, a mixture of inner mitochondrial membrane proteins, or a mixture of erythrocyte membrane proteins. The method involves (1) saturating a small column of phenyl-Sepharose (1-2 mL) with one of the alkyl detergents at a pH of 8 or 9 and an ionic strength of 0.01, (2) applying a detergent-solubilized membrane protein sample containing as much as 20 mg/mL of Triton X-100, Triton N-101, or Nonidet P-40, and (3) eluting the protein with buffer containing the detergent with which the resin had been saturated. With this approach, 90-99% of the detergent in the initial protein sample was exchanged for the second detergent with an 80-100% recovery of protein. The advantages of this method over previous approaches for exchanging detergents include the rapidity of the technique and the apparent general applicability of the method to a wide variety of detergents and membrane proteins.

Animals↗
PubMed1989

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles.

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs.

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl beta-maltoside (BrDM) or 10,11-dibromoundecanoyl beta-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I(-)), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.

Biophysical Phenomena↗
PubMed2003

Insights into the association of FcgammaRII and TCR with detergent-resistant membrane domains: isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution.

Plasma membrane rafts are routinely isolated as detergent-resistant membranes (DRMs) floating in detergent-free density gradients. Here we show that both the presence and exclusion of TX-100 during the density gradient fractionation have profound effects on the location of FcgammaRII and TCR in DRM fractions. The presence of TX-100 during fractionation promoted solubilization of non-cross-linked FcgammaRII when the receptor was insufficiently dissolved upon cell lysis. In the detergent-supplemented gradients, TX-100 micelles floated, further enhancing dissociation of FcgammaRII and TCR from DRMs and promoting a shift of the receptors toward higher-density fractions. Hence, fractionation of cell lysates over the detergent-containing gradients enables isolation of DRMs devoid of weakly associated proteins, like nonactivated FcgammaRII and TCR. On the other hand, in a detergent-free gradient, non-cross-linked FcgammaRII, fully soluble in 0.2% TX-100, was recovered in DRM fractions. Moreover, employment of the TX-100-free gradient for refractionation of intermediate-density fractions, derived from detergent-supplemented gradients and containing FcgammaRII and TCR, resulted in flotation of the receptors to buoyant fractions. An analysis of the TX-100 concentration revealed that after fractionation of 0.2% TX-100 cell lysates in the absence of detergent, the level of TX-100 in DRM fractions was reduced to 0.01%, below the critical micelle concentration. Therefore, fractionation of detergent cell lysates over detergent-free gradients can mimic conditions for a membrane reconstitution, evoking association of a distinct subset of membrane proteins, including FcgammaRII and TCR, with DRMs.

Cell Membrane↗
PubMed1987

Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent.

We have established the generality of using detergents for facilitating the reactivation of 6 M guanidinium chloride-denatured rhodanese that was recently described for the nonionic detergent lauryl maltoside (LM) (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). We report here that not only LM but other nonionic as well as ionic and zwitterionic detergents also have favorable effects in reactivating the denatured enzyme. Not all detergents are useful, and the favorable effects occur over a limited concentration range. Above and below that range there is little or no effect. Zwittergents, which represent a homologous series with varying critical micelle concentrations (CMCs) are effective only above their CMCs. Induction phases occur in the progress curves of rhodanese refolded in the presence of the effective detergents, suggesting the presence of refolding intermediates that are apparently stabilized by detergent interactions. Gel filtration chromatography of rhodanese with and without LM suggests that even though the renaturation of the denatured enzyme requires detergent at concentrations above its CMC, the enzyme does not bind an amount of detergent equivalent to a micelle. It is suggested that renaturation of other proteins might also be assisted by inclusion of "nondenaturing" detergents, although the optimal conditions will have to be determined for each individual case.

Detergents↗
PubMed1997

The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.

The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.

Benzoquinones↗
PubMed1998

Detergent extraction of herpes simplex virus type 1 glycoprotein D by zwitterionic and non-ionic detergents and purification by ion-exchange high-performance liquid chromatography.

Detergents (surfactants) are the key reagents in the extraction and purification of integral membrane proteins. Zwitterionic and non-ionic detergents were used for the extraction of recombinant glycoprotein D (gD-1) of herpes simplex virus type 1 (HSV-1) from insect cells infected with recombinant baculovirus. The highest yield was obtained with the two alkyl carboxybetaine detergents (N-dodecyl-N,N-dimethylammonio)undecanoate [DDMAU, critical micelle concentration (CMC) = 0.13 mM] and (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM). Therefore these zwitterionic detergents were used as additives to the elution buffers in ion-exchange high-performance liquid chromatography (HPIEC) to purify gD-1 of HSV-1 from the extracts. The non-ionic detergent pentaethyleneglycol monodecyl ether (C10E5) that was used in earlier studies [R.A. Damhof, M. Feijlbrief, S. Welling-Wester, G.W. Welling, J. Chromatogr. A, 676 (1994) 43] was used for comparison. Two columns were used, Mono Q and Resource Q, at 1 and 5 ml/min flow-rates, respectively. The results show that the detergents DDMAU and C10E5 are superior to DDMAB, when the detergents were used as additives to the elution buffers at 0.2% (w/v). With 0.2% DDMAB in the eluent, purification of HSV gD-1 was not possible. Detergents with a high CMC may be less suitable as additives in elution buffers. HPIEC at flow-rates of 1 and at 5 ml/min showed satisfactory results. At 5 ml/min HSV gD-1 was mainly concentrated in two eluent fractions. The highest recovery of gD-1 was obtained either by chromatography of a C10E5 extract using a Mono Q column at a flow-rate of 1 ml/min or by chromatography of a DDMAU extract using a Resource Q column at a flow-rate of 5 ml/min.

Animals↗
PubMed1989

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction.

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Toxicity of laundry detergent components to a freshwater cladoceran and their contribution to detergent toxicity.

The toxicity of 39 laundry detergent components including surfactants, enzymes, builders, fabric brighteners, fillers, and coloring agents to the cladoceran Ceriodaphnia cf. dubia was determined. The difference between the most and the least toxic components was approximately 17,000-fold and 1,000,000-fold for the mg/L and mmol/L EC50 data, respectively. Two of the components had high toxicity (EC50 values < 1 mg/L), 11 moderate toxicity (EC50 values between 1 and 10 mg/L), and the remaining 26 components had low toxicity (EC50 values > 10 mg/L). Analysis revealed that mixtures of the components interacted antagonistically, additively, and synergistically. On a molarity basis the most toxic group of compounds was the surfactants followed by the brighteners. The most toxic individual components included sodium carboxymethyl cellulose, sodium silicate solution, four brighteners, sodium perborate tetrahydrate, and the surfactants. Many of the most toxic components, however, contributed very little to the toxicity of the detergents due to being present in the detergents at low concentrations. The main contributors to the toxicity of detergents were the sodium silicate solution and the surfactants-with the remainder of the components contributing very little to detergent toxicity. The potential for acute aquatic toxic effects due to the release of secondary or tertiary sewage effluents containing the breakdown products of laundry detergents may frequently be low. However, untreated or primary treated effluents containing detergents may pose a problem. Chronic and/or other sublethal effects that were not examined in this study may also pose a problem.

Animals↗
PubMed1991

Structure of the detergent phase and protein-detergent interactions in crystals of the wild-type (strain Y) Rhodobacter sphaeroides photochemical reaction center.

Rhodobacter sphaeroides (strain Y) reaction center (RC) crystals were grown in the presence of n-octyl beta-glucoside (beta-OG). In order to determine the structure of the detergent phase in these crystals, low-resolution neutron diffraction experiments were performed at different contrasts obtained by varying the H2O/D2O ratio in the solvent. From the contrast variation data and from the RC atomic coordinates determined by X-ray diffraction [Arnoux, B., Ducruix, A., Reiss-Husson, F., Lutz, M., Norris, J., Schiffer, M., & Chang, C. H. (1989) FEBS Lett. 258, 47-50], a model was obtained for the structure of the detergent phase in the crystal. The detergent forms a ring-shaped micelle surrounding the most hydrophobic part of the transmembrane alpha helices of the RC. Each detergent ring is connected to two next-neighbor rings by intermicellar bridges. The detergent phase is organized thus in infinite zigzag chains parallel to the b axis of the P2(1)2(1)2(1) unit cell. The main interactions between beta-OG molecules and the RC molecules are hydrophobic and are localized at the level of the transmembrane alpha helices. This interaction replaces the phospholipid-protein interaction existing in vivo in the membrane and, to some extent, also the light harvesting complex-protein interaction. Secondary hydrophilic interactions are found between a few of the charged residues of the H subunit and the hydrophilic surface of the detergent ring from a neighboring RC molecule. A comparison with a previous study on Rhodopseudomonas viridis crystals [which grow in the presence of lauryldimethylamine N-oxide (LDAO) and belong to a different space group] [Roth, M., Lewit-Bentley, A., Michel, H., Deisenhofer, J., Huber, R., & Oesterhelt, D. (1989) Nature 340, 659-661] shows a quasi identity of shape and position of the beta-OG and LDAO rings around the transmembrane alpha helices. The secondary interactions, involving in both cases the external surface of the H subunit, differ because of the different molecular packing in the two space groups. The role and structural requirements of the detergent in the crystallization process are discussed.

Algorithms↗
PubMed1977

Effect of polyol detergents on cholesterol and triglyceride absorption. Hypolipidemic action of chronic administration of hydrophobic detergent.

Nonionic detergents were found to affect absorption of neutral lipid and cholesterol. The effects of these detergents were related to their physicochemical properties. The most effective detergents for producing an inhibition of lipid absorption were detergents containing 90% hydrophobic components. Hydrophilic detergents did not interfere with lipid absorption and in some cases, it was facilitated. Studies done over a period of one month demonstrate that addition of a hydrophobic detergent to a high fat-high cholesterol diet results in lower serum levels of cholesterol and triglyceride and prevents cholesterol accumulation in the liver of the rat. This effect was also associated with significant loss of body weight.

Animals↗
PubMed1985

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes.

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.

Animals↗
PubMed1976

Determination of molecular weight of the protein moiety in protein-detergent complexes without direct knowledge of detergent binding.

Sedimentation equilibrium measurements can be used to determine the molecular weight of the protein moiety of a protein-detergent complex without prior knowledge of detergent binding. The procedure is to adjust the solvent density by addition of D2O so as to blank out the contribution of bound detergent to the sedimentation potential. An approximate measure of detergent binding can be obtained from the effect of solvent density on the sedimentation result. The procedure is also applicable to protein-lipid complexes. It can be used for complexes containing both lipid and detergent if the lipid content is known. The use of the method is demonstrated by experimental data for the AI polypeptide of serum high density lipoprotein, in separate complexes with nonionic detergents and with a phospholipid.

Apolipoproteins↗
PubMed1997

On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes.

Detergent-insoluble membrane fragments that are rich in sphingolipid and cholesterol can be isolated from both cell lysates and model membranes. We have proposed that these arise from membranes that are in the liquid-ordered phase both in vivo and in vitro [Schroeder et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 12130-12134]. In order to detect formation of the liquid-ordered phase while avoiding possible detergent artifacts, we have now used fluorescence quenching to examine the phase behavior of mixtures of phosphatidylcholines, sphingolipids, and cholesterol. Phase separation was found in binary mixtures of either dipalmitoylphosphatidylcholine (DPPC) or sphingomyelin (SM) and a nitroxide-labeled phosphatidylcholine (12SLPC). A DPPC- or SM-enriched solidlike gel phase coexisted with a 12SLPC-enriched liquid-disordered fluid phase at 23 degrees C. As expected, phase separation was not seen at low concentrations of DPPC or SM. Instead, only a uniform fluid phase was present. Including 33 mol % cholesterol in model membranes greatly promoted phase separation. Phase separation was seen at higher temperatures and/or at lower concentrations of DPPC or SM in the presence of cholesterol than in its absence. Mixtures of DPPC or SM and cholesterol are known to form the liquid-ordered phase. Therefore, the fact that phase separation was observed in the cholesterol-containing membranes shows that liquid-ordered and liquid-disordered phase domains coexist. At 37 degrees C, the SM-enriched liquid-ordered phase was first seen at a SM/PC ratio of close to 0.25, when SM made up 17% of the total lipid including cholesterol. (This is similar to or less than the SM concentration of the plasma membranes of mammalian cells.) Furthermore, the detergent insolubility of cholesterol-containing model membranes correlated well with the amount of liquid-ordered phase as detected by fluorescence quenching. Thus, the detergent-insoluble membranes isolated from cells are likely to exist in the liquid-ordered phase prior to detergent extraction. The promotion of liquid-ordered phase formation may be an important function of cholesterol and sphingolipids in cells and may be a major distinction between the cholesterol- and sphingolipid-rich plasma membrane and most other cellular membranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.

Bacterial Outer Membrane Proteins↗
PubMed1997

Correlation of acid detergent lignin and Klason lignin with digestibility of forage dry matter and neutral detergent fiber.

The acid detergent lignin and Klason lignin methods were compared for their correlation with forage digestibility. Thirty-six forages, including C3 legumes and C3 and C4 grasses, were analyzed for sulfuric acid detergent lignin, Klason lignin, and in vitro digestibilities of dry matter (DM) and neutral detergent fiber (NDF). Twenty of these forages were also fed to lambs at restricted intake for measurement of DM and NDF digestibilities. Lignin concentrations determined by the two lignin methods were positively correlated, and the Klason lignin value was always greater than the acid detergent lignin concentration. The largest differences were observed for grass forages. Digestibilities of forage DM and NDF were negatively correlated with both lignin methods for the in vitro system and the lamb digestibility trials. The degree of correlation for the two lignin methods with digestibility was generally similar across all forages and within forage classes. Slopes of linear regressions of digestibility on lignin concentration did not differ between legumes and grasses. Although the sulfuric acid detergent lignin and Klason lignin procedures gave very different estimates of forage lignin concentration, they were similarly correlated with digestibility and should yield predictions of forage digestibility that have similar accuracy.

Animal Feed↗