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

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

Toxicity of synthetic detergents to fish and aquatic invertebrates

Synthetic detergents are reported to be acutely toxic to fish in concentrations between 0.4 and 40 mg/1. Factors affecting toxicity include the molecular structure of the detergent, water hardness, temperature and dissolved oxygen concentration; the age and species of the test fish, and acclimation to low concentrations of detergent. Some of these factors appear to be of only limited importance. Gill damage is the most obvious acute toxic effect; the immediate cause of death may be asphyxiation, but detergents may also be toxic internally. Lethal effects not related to gill damage have not been investigated. Sublethal effects include retardation of growth, alteration of feeding behaviour and inhibition of chemoreceptor organs. Low levels of detergents may also increase the uptake of other pollutants. Invertebrates, especially in their juvenile stages, are extremely sensitive to detergents: concentrations below 0.1 mg/1 interfere with growth and development in some species. The interactions between detergents and proteins, and their influence on membrane permeability may be the basis of the biological action of detergents. Detergents in natural waters are usually partially degraded, and a maximum permissible concentration of 0.5 mg/1 would probably be harmless under most conditions.     

Destabilization of collagen structure by amides and detergents in solution

The effects of amides and detergents on collagen to gelatin transition have been studied at neutral pH. Simple amides denature the protein. The substitution of H-atoms by the alkyl groups at the nonpolar end of amide increases the effectiveness of the compounds in destabilizing the collagen structure whereas substitution of the H-atom at the polar amide end shows marginal effects on the collagen transition. The capabilities of these reagents to denature collagen are much less pronounced than their effects on denaturing globular proteins. Anionic detergents are found to destabilize collagen at very low concentrations (below their cmc values). In this respect, the effects of the detergents on collagen are comparable to the denaturing effects of the detergents on globular proteins. The effect of detergents increases with the increase in the length of the alkyl chain. The structure of the anion in the detergent is also important as seen from the lower potency of the sulfonate containing detergent compared to the sulfate containing detergent in denaturing collagen. Cationic and nonionic detergents do not denature collagen.     

STUDIES ON THE PHYSIOLOGICAL EFFECTS OF NON-POLAR-POLAR ORGANIC ELECTROLYTES : I. THE INFLUENCE UPON THE RESTING POTENTIAL OF FROG MUSCLE

1. The commonly used detergents have a poisonous effect, which is due to the non-polar-polar configuration of their organic anion. The non-polar organophilic half of the ion is built up by a long chain of alkyl radicals (8 to 18 carbon atoms), the polar hydrophilic half by a sulfonate or sulfate. If brought into contact with the organic surface membrane of a cell, this structure, due to the strong attachment of the alkyl chains to its surface, and due to the pull of the hydrophilic part towards the surrounding water, is subjected to a heavy stress terminating in tearing to pieces the membrane (by denaturing and loosening the membrane components; bacteriolysis, cytolysis). Correspondingly, with frog muscle, one end of which has been treated with the detergent solution, an irreversible negative injury potential is produced. 2. Applying, instead, the compounds bearing short chains of alkyl radicals (1 to 6 carbon atoms), producing less stress on the membrane and correspondingly a slighter derangement of its architecture, a reversible positive resting potential appears. This is interpreted to be the effect of the non-polar part of the anion, which, due to its surface activity, intrudes into the pores of the membranes, notwithstanding the negative charge of their walls. 3. The short chained detergents seem to be replaceable by various organic "semidetergents," the organophilic behavior of their anion being represented by a slight chemical affinity (NH₂), the hydrophilic by the effect of a carboxyl group (COO) instead of sulfate or sulfonate. The effect of the semidetergents on muscle is a positive reversible potential. Their physiological significance may be visualized as a functional activation.     

Studies on the physiological effects of non-polar-polar organic electrolytes; the influence of detergents upon the potentiometric reaction and the contractility of nerve and muscle

In a previous paper it has been shown that the nonpolar-polar anionic detergents can be divided into two main groups. One chemically characterized by a relatively long chain of non-polar alkyl groups, which in solution are in contact with one end of a muscle and, locally adhering to it, produce a permanent negative injury potential. This is generally accompanied by a loss of excitability. The second group, distinguished by a relatively short chain of non-polar alkyl groups acts reversibly, ordinarily preserves the excitability and, in contrast to the first group, produces locally a reverse positive potential. For reasons mentioned before, this appears likely to correspond to an increased activity. These concepts have been tested in this second paper. The measurements of the resting potentials of muscles have been supplemented by measurements on frog sciatics with the result that there are brought about, again by the detergents with long alkyl chains, regular irreversible negative resting potentials and with the short chain compounds reversible positive potentials are aroused. Furthermore, in addition there appeared the hardly expected result that muscle stimulated in the presence of short chain detergents responded with an even higher contraction. We have endeavored to explain this on the basis of general considerations concerning the physical chemistry of the excitatory process. More direct evidence of this rise of excitability under the influence of the short chain non-polar-polar detergents will be presented in the next papers on studies concerning chronaximetric measurements on nerve, referring particularly to the semidetergents, and concerning the effects of detergents in general upon the heart beat of a clam.     

INFLUENCE OF CELL WALL COMPOSITION ON THE RESISTANCE OF TWO CHLORELLA SPECIES (CHLOROPHYTA) TO DETERGENTS1

The influence of dodecylbenzene sulfonate (DBS) and Triton X-100 (TX-100) was examined on two species of Chlorella exhibiting conspicuous differences in cell wall composition. Chlorella emersonii has both a classical polysaccharidic wall and a thin trilaminar outer wall (TLS) composed of nonhydrolyzable macromolecules. Chlorella vulgaris lacks a TLS. Photosynthetic capacity was measured following short exposures (1 h) of the algae at different physiological stages to high DBS and TX-100 concentrations, up to 1 g·L^?1. Comparisons with untreated controls indicated that 1) the presence of a TLS in C. emersonii was associated with a very high resistance to the anionic (DBS) and nonionic (TX-100) detergents at all growth stages, and net photosynthesis was not significantly affected in that species, 2) a high toxicity, particularly pronounced with TX-100, was observed for actively growing cells of the TLS-devoid species, C. vulgaris, and 3) aging exerted a protective influence, especially efficient against DBS, on the latter species. Additional observations, including fluorescence spectra and high-performance liquid chromatography pigment analyses, were conducted following short exposures of actively growing cells. Fluorescence emission spectra revealed that the chlorophyll a-protein complexes in thylakoid membranes were not substantially affected by DBS and TX-100, even in the case of C. vulgaris. In sharp contrast, fluorescence excitation spectra on the latter species showed 1) that excitation transfer from antenna pigments to chlorophyll a in reaction centers was substantially altered with both detergents and 2) that the two detergents affected different parts of the photosynthetic system of the TLS-devoid species. Analyses of C. vulgaris extracts indicated significant decreases in pigment content following exposure to DBS and, to a lesser extent, to TX-100. Longer exposure experiments (1 day) were conducted with actively growing algae. The TLS-containing species still showed a very high resistance and no important changes in photosynthetic capacity compared to cells exposed for 1 h. For the sensitive TLS-devoid species, the detrimental influence of TX-100, already very high after 1 h, was not increased. DBS toxicity was markedly increased and may reflect a lower uptake rate of DBS by C. vulgaris. Taken together, these observations confirm the important protective role of TLS against detergents. They also provide information on the factors controlling detergent toxicity in the sensitive, TLS-devoid species and on the different modes of action of DBS and TX-100 on its photosynthetic system. Such large differences in microalgal sensitivity to detergents, related to TLS occurrence, should have important consequences for the selection of suitable species in toxicity tests.     

N1N8-bis(gamma-glutamyl)spermidine cross-linking in epidermal-cell envelopes. Comparison of cross-link levels in normal and psoriatic cell envelopes.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Dodecyl Maltoside Protects Membrane Proteins In Vacuo

Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a β-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The β-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an α-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.     

Detergents inhibit exocytosis in PC 12 cells: evidence for an effect on ion fluxes

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+-stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X-100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 microM and 10 microM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4). The inhibitory effect of 30 microM Triton X-100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X-100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X-100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X-100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13-14 degrees C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished. In the presence of 30 microM Triton X-100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50-60%. These data suggest that the site of action of Triton X-100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.     

INFLUENCE OF FLUCTUATING PHOSPHATE SUPPLY ON THE REGULATION OF PHOSPHATE UPTAKE BY THE BLUE-GREEN ALGA ANACYSTZS NIDULANS1

The blue-green alga Anacystis nidulans Drouet (Synechococcus leopoliensis Raciborski) cultivated under phosphate-limited conditions adopts a threshold value in the nanomolar range below which uptake ceases. In this study, we investigated the influence of phosphate pulses on the regulation of uptake behavior during reestablishment of the threshold value. Short-term pulses had only a slight effect on uptake kinetics and, hence, on the threshold value, even if the population had been exposed several times to elevated concentrations above the steady-state level in the growth medium. The threshold value was also practically insensitive to the amount of phosphate stored during these short-term fluctuations. Long-term phosphate pulses resulted in a transition to a metastable state that was characterized by a severalfold higher threshold value. This transition, apparently an adaptation to the transiently elevated phosphate concentrations, was further studied by following the influx of ³²P-phosphate at constant external concentrations and was shown to be complete after a period of 10–20 min. After adaptation to pulses, the uptake behavior followed a linear flow-force relation over a wide range of external concentrations. This behavior was explained by the simultaneous operation of at least two uptake systems with different, but coordinated kinetic parameters. This linear flow-force relation facilitated a direct determination of the threshold value from uptake measurements. For applicability in the field the force-flow relation can be a diagnostic tool to assay for fluctuating phosphate and to establish threshold values below the normal measurable range .     

Dodecyl Maltoside Protects Membrane Proteins In Vacuo

Molecular dynamics simulations have been used to characterize the effects of transfer from aqueous solution to a vacuum to inform our understanding of mass spectrometry of membrane-protein-detergent complexes. We compared two membrane protein architectures (an α-helical bundle versus a β-barrel) and two different detergent types (phosphocholines versus an alkyl sugar) with respect to protein stability and detergent packing. The β-barrel membrane protein remained stable as a protein-detergent complex in vacuum. Zwitterionic detergents formed conformationally destabilizing interactions with an α-helical membrane protein after detergent micelle inversion driven by dehydration in vacuum. In contrast, a nonionic alkyl sugar detergent resisted micelle inversion, maintaining the solution-phase conformation of the protein. This helps to explain the relative stability of membrane proteins in the presence of alkyl sugar detergents such as dodecyl maltoside.     

Toxic action of several lethal concentrations of an anionic detergent on the gills of the brown trout (Salmo trutta L.)

The pathological effects often lethal concentrations of the anionic detergent, sodium lauryl sulphate, on the gills of Salmo trutta L. have been studied by light and electron microscopy. At concentrations to 120 mg/1 (medium survival times >1 h), epithelial cell death is associated with lysosome formation. Acute inflammation of the gill tissue, extensive detachment of the epithelium and, except at the lowest concentrations, collapse of the pillar cell system occur. At concentrations above 120 mg/1 (medium survival times <1 h) very rapid lysis of cells results in the complete disruption of cellular and tissue structure. Changes in the gross structure of the gills are explainable in terms of the rate and nature of toxic action at the cellular level. Review of the biomedical literature suggests the observed effects of sodium lauryl sulphate on gill cells correspond to the two mechanisms by which detergents cause death in isolated cells. These are autolysis, i.e. lysis by the action of the cell's own enzymes, induced by an initial lesion in the cell membrane whose precise nature is not known; and rapid lysis by the direct action of the detergent on the cell constituents.     

Detergents as probes of reconstituted rat liver cytochrome P-450 function

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.     

A gas chromatography–mass spectrometry method to monitor detergents removal from a membrane protein sample

In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.     

Lipopeptide detergents designed for the structural study of membrane proteins

The structural study of membrane proteins requires detergents that can effectively mimic lipid bilayers, and the choice of detergent is often a compromise between detergents that promote protein stability and detergents that form small micelles. We describe lipopeptide detergents (LPDs), a new class of amphiphile consisting of a peptide scaffold that supports two alkyl chains, one anchored to each end of an alpha-helix. The goal was to design a molecule that could self-assemble into a cylindrical micelle with a rigid outer hydrophilic shell surrounding an inner lipidic core. Consistent with this design, LPDs self-assemble into small micelles, can disperse phospholipid membranes, and are gentle, nondenaturing detergents that preserve the structure of the membrane proteins in solution for extended periods of time. The LPD design allows for a membrane-like packing of the alkyl chains in the core of the molecular assemblies, possibly explaining their superior properties relative to traditional detergents in stabilizing membrane protein structures.     

THE INFLUENCE OF MINIMAL NARCOTIC DOSES ON THE RESPIRATION OF ERYTHROCYTES

Low concentrations of ethyl alcohol stimulate the respiration of mammalian erythrocytes in vitro. Low concentrations of ethyl urethan remain without effect on, or tend slightly towards depressing the respiration of mammalian erythrocytes in vitro. It is suggested that this may be due to the oxidizable nature of alcohol, and the non-oxidizable nature of urethan, properties which come into evidence only when these narcotics are present in such low concentrations that the threshold of inhibition (narcosis) has not been reached.     

Comparative studies of the artificial chaperone-assisted refolding of thermally denatured bovine carbonic anhydrase using different capturing ionic detergents and beta-cyclodextrin

Artificial chaperone-assisted refolding has been shown to be an effective approach for improving the refolding yield of some of the denatured proteins. Since identical concentrations of various detergents do not induce similar variations in the protein structures, we arranged to evaluate the artificial chaperoning capabilities of several ionic detergents as a function of charge, structure, and the hydrophobic tail length of the detergent. Our results indicate that carbonic anhydrase can be refolded from its denatured state via artificial chaperone strategy using both anionic and cationic detergents. However, the extent of refolding assistance (kinetic and refolding yield) were different due to protein and detergent net charges, detergent concentrations, and the length of hydrophobic portion of each detergent. These observed differences were attributed to physical properties of CA-detergent complexes and/or to the kinetics of detergent stripping by beta-cyclodextrin from the protein-detergent complexes which is apparently dependent on the detergent-beta-CD association constants and the nature of the partially stripped complexes.     

Detergents for the stabilization and crystallization of membrane proteins

The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult to maintain a solubilized membrane protein in a native conformational state, and the non-specific aggregation of detergent-solubilized proteins is a common problem. Empirical "stability screens" can be helpful in choosing which detergents, and which detergent concentrations, may be optimal for a given system.     

Non-lytic, non-ionic detergent extractions of plasma membrane constituents from normal and transformed fibroblasts

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cells employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, ⁵¹Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodina- tion followed by detergent extraction permits demonstration of a 100 000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.