Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.     

Detergent modification of myosin function and structure in solution

Rabbit skeletal muscle myosin and myosin subfragment 1 (S1) MgATPase activities were increased 2-3-fold by the addition of a variety of molecules that contained single straight saturated 12-16-carbon chains. The nonionic detergent dodecyl nonaoxyethylene ether (C12E9) increased the activity of S1 to 50% of maximum at a free C12E9 concentration of 27 +/- 9 microM. The activation was reversible and was not due to chemical modification of S1 amino acid side chains. The Vmax for actin-activated S1 MgATPase activity was increased 3-fold by C12E9. The apparent association constant for S1 binding to pure F-actin was reduced 3-fold by C12E9. The [C12E9] dependencies of the increase in S1 and acto-S1 MgATPase activities and of the decrease in acto-S1 binding were equal, within experimental uncertainty, suggesting that a single detergent-induced S1 conformational change is sufficient to explain the results. The stoichiometry of C12E9 bound to S1 in the S1-C12E9 complex was estimated, by the S1 concentration dependence of the C12E9 activation midpoint and by the light-scattering increase when S1 and detergent were mixed, to be 7 and 57 C12E9 molecules per S1, respectively. The results are discussed in relation to possible structural aspects of the mechanism of action for S1 and acto-S1 MgATPase activities.     

Modulatory effect of two cardioglycosides on reconstituted Na+/K(+)-ATPase in proteoliposomes.

1. Na,K-ATPase was extracted from Cavia cobaya kidneys, solubilized with nonionic detergent C12E8 (octaethyleneglycol dodecyl monoether) in mixed lipid-detergent-protein micelles. The Na,K-ATPase specific activity was 30-35 IU/mg protein. 2. The enzyme was reconstituted in vesicles, made of phosphatidylethanolamine and cholesterol: an enhancement of +60% in specific activity was obtained. 3. Two different vesicle-types were carried out: open liposomes (partially organized membranes) and closed liposomes. 4. Proteoliposomes were employed for measuring the modulatory effect of two cardioglycosides: ouabain and digoxin. 5. Inhibition of the Na,K-ATPase activity revealed apparent Ki of 1.25 microM for ouabain and 0.25 microM for digoxin in open liposomes, and apparent Ki of 0.75 microM for ouabain and of 1.75 microM for digoxin in closed liposomes. 6. Maximum enhancement of enzymatic activity was found at concentrations of 5-0.5 nM for ouabain and 5-1 nM for digoxin in open liposomes, and 25-1 nM for both digoxin and ouabain in closed liposomes.     

Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

3-Hydroxy-3-methylglutaryl-CoA reductase (NADPH) was solubilized with polyoxyethylene ether (Brij) W-1 from a heavy-membrane fraction, sedimented at 16000 X g from a cell-free homogenate of four-day-old, dark-grown radish seedlings (Raphanus sativus L.). Approximately 350-fold purification of the solubilized enzyme activity was achieved by (NH4)2SO4 precipitation followed by column chromatography on DEAE-Sephadex A-50, blue-dextran-agarose and HMG-CoA-hexane-agarose. The presence of detergent, which was required at all times to maintain activity, did not interfere with the chromatographic procedures used. Sucrose density centrifugation suggested an apparent molecular mass of 180 kDa with subunits of 45 kDa (polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate). The enzyme was stable at 67.5 degrees C for 30 min in the presence of glycerol, dithioerythritol and detergent. Studies of enzyme stability and activation indicate that the enzyme is a hydrophobic protein with free thiol groups that are essential for full activity. The activation energy was estimated to be 92 kJ (Arrhenius plot). Antibodies raised against rat liver and yeast hydroxymethylglutaryl-CoA (HMG-CoA) reductase failed to bind or inactivate the radish enzyme. When both HMG-CoA and NADPH concentrations were varied, intersecting patterns were obtained with double-reciprocal plots. The apparent Km values determined in this way are 1.5 microM [(S)-HMG-CoA], and 27 microM (NADPH). Concentrations of NADPH greater than 150 microM caused substrate inhibition at low HMG-CoA concentrations resulting in deviations from linearity in secondary plots. Analysis of these data and the product inhibition pattern suggest a sequential mechanism for the reduction of HMG-CoA to mevalonic acid with HMG-CoA being the first substrate binding to the enzyme, followed by NADPH.     

The polar headgroup of the detergent governs the accessibility to water of tryptophan octyl ester in host micelles

Many attempts have been made to rationalize the use of detergents for membrane protein studies [J. Biol. Chem. 264 (1989) 4907]. The barrier properties of the detergent headgroup may be one parameter critically involved in protein protection. In this paper, we analyzed these properties using a model system, by comparing the accessibility of tryptophan octyl ester (TOE) to water-soluble collisional quenchers (iodide and acrylamide) in three detergent micelles. The detergents used differed only in the chemical nature of their polar headgroups, zwitterionic for dodecylphosphocholine (DPC) and nonionic for octa(ethylene glycol) dodecyl monoether (C(12)E(8)) and dodecylmaltoside (DM). In all cases, in phosphate buffer at pH 7.5, the binding of 5 microM TOE was complete in the presence of a slight excess of detergent micelles over TOE molecules, resulting in a significant blue shift and greater intensity of TOE fluorescence emission. The resulting quantum yield of bound TOE was between 0.08 (in DPC) and 0.12 (in DM) with an emission maximum (lambda(max)) of approximately 335 nm whatever the detergent micelle. Time-resolved fluorescence intensity decays of TOE at lambda(max) were heterogeneous in all micelles (3-4 lifetime populations), with mean lifetimes of 1.7 ns in DPC, and 2 ns in both C(12)E(8) and DM. TOE fluorescence quenching by iodide, in detergent micelles, yielded linear Stern-Volmer plots characteristic of a dynamic quenching process. The accessibility of TOE to this ion was the greatest with C(12)E(8), followed by DPC and finally DM (Stern-Volmer quenching constants K(sv) of 2 to 5.5 M(-1)). In contrast, the accessibility of TOE to acrylamide was greatest with DPC, followed by C(12)E(8) and finally DM (K(sv)=2.7-7.1 M(-1)). TOE also presents less rotational mobility in DM than in the other two detergents, as shown from anisotropy decay measurements. These results, together with previous TOE quenching measurements with brominated detergents [Biophys. J. 77 (1999) 3071] provide reference data for analyzing Trp characteristics in peptide (and more indirectly protein)-detergent complexes. The main finding of this study was that TOE was less accessible (to soluble quenchers) in DM than in DPC and C(12)E(8), the cohesion of DM headgroup region being suggested to play a role in the ability of this detergent to protect function and stability of solubilized membrane proteins.     

Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland.

Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.     

Soluble and enzymatically stable (Na+ + K+)-ATPase from mammalian kidney consisting predominantly of protomer alpha beta-units. Preparation, assay and reconstitution of active Na+, K+ transport

Soluble (Na+ + K+)-ATPase consisting predominantly of alpha beta-units with Mr below 170 000 was prepared by incubating pure membrane-bound (Na+ + K+)-ATPase (35-48 mumol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). (Na+ + K+)-ATPase and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5-3, at which 50-70% of the membrane protein was solubilized. The soluble protomeric (Na+ + K+)-ATPase was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5-6, at the expense of partial inactivation, but (Na+ + K+)-ATPase and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, S20,w was 7.4 S for the fully active (Na+ + K+)-ATPase, 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000-170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active (alpha beta)2-dimers or (alpha beta)3-trimers with S20,w = 10-12 S and apparent molecular masses in the range 273 000-386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles.     

Effects of the incorporation of CHAPS into SDS micelles on neuropeptide-micelle binding: separation of the role of electrostatic interactions from hydrophobic interactions

It is well known that neuropeptides interact with lipid vesicles in a manner similar to biological membranes, with electrostatic interactions between the two providing a mechanism for concentrating the peptide at the vesicle's surface, followed by hydrophobic interactions between the peptide and the core of the vesicle that induce and stabilize secondary structure motifs. In an effort to understand these interactions to a greater extent, our group has developed a series of anionic micelles (SDS) containing various concentrations of the bile salt CHAPS, which is used as a model for cholesterol. The incorporation of CHAPS into the hydrophobic core of these micelles should alter the degree to which the neuropeptide can insert itself, affecting structure. These interactions were investigated using two-dimensional NMR, pulse-field gradient (PFG) NMR, and molecular modeling experiments. The results of this study clearly indicate that electrostatic and hydrophobic interactions between the micelle and neuropeptide are completely independent of one another. Increasing the concentration of CHAPS to 15 mM in the micelles blocks the insertion of the hydrophobic side chains of the neuropeptide into the hydrophobic core of the micelles. The electrostatic interactions as determined by diffusion measurements are not affected by the presence of increasing CHAPS concentration. Our observations are consistent with the predictions of Seelig (A. Seelig and J. Seelig, "Interaction of Drugs and Peptides with the Lipid Membrane," in Structure and Function of 7TM Receptors, T. W. Schwartz, S. A. Hjorth, and T. S. Kastrup, Eds., Munksgaard: Location, 1996).     

Detergent solubilization of the interleukin 1 receptor

Interleukin 1 (IL 1) receptors were solubilized from membranes prepared from murine EL-4 thymoma cells with the zwitterionic detergent 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Binding of IL 1 to the solubilized receptor was detected by a polyethylene glycol (PEG) precipitation procedure. Concentrations of CHAPS from 4 to 8 mM were effective in solubilizing the IL 1 receptor. At 10 mM CHAPS, there was some loss in binding activity, whereas 2 mM CHAPS was completely ineffective in solubilizing the receptor. Detergent concentrations of 4 mM were routinely used. The solubilized receptor retains the ability to bind 125I-IL 1 in a specific and saturable manner. Scatchard analysis reveals a single type of high affinity binding site having an apparent dissociation constant (KD) of approximately 1.2 X 10(-10) M. Nearly identical KD values are observed for membrane fractions. There are approximately 400 to 500 fmol receptor/mg protein in the detergent extract, corresponding to a two- to threefold enrichment in the Bmax observed for membranes. There is no loss in receptor activity as determined by complete recovery of the total number of binding sites from membranes after solubilization. Binding kinetics show that apparent steady state for the solubilized receptor is reached after 60 min at 37 degrees C. The binding of 125I-IL 1 is essentially irreversible because relatively little bound ligand can be dissociated from the receptor on the addition of excess unlabeled IL 1 at 37 degrees C. Both human IL 1 alpha and IL 1 beta compete for binding of 125I-IL 1 to the soluble receptor, confirming that IL 1 alpha and IL 1 beta bind to the same receptor. Other recombinant proteins, including interferon-alpha A, interferon-gamma, and interleukin 2 have no inhibitory effect.     

Oligomeric states of the detergent-solubilized human serum paraoxonase (PON1)

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C(12)E(8) micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.     

Inhibition of soybean lipoxygenase-1 by n-alcohols and n-alkylthiols.

A series of n-alcohols and n-alkylthiols with carbon chains from 2 to 12 were examined for the inhibition of soybean lipoxygenase-1 (L-1). The alcohol produces a competitive inhibition, the extent of which increases with an increase in the carbon number of alkyl chain up to 8. Whereas the inhibition of the alkylthiol is noncompetitive, the extent of which is almost independent from the carbon number. From the behavior of pKi dependence on the carbon number of the alcohol, the decyl group appears to be optimum to bind to L-1. The thermodynamic analysis for the inhibition based upon van 't Hoff equation indicates positive enthalpy and entropy changes for the binding of the alcohol to the enzyme and negative enthalpy and positive to negative entropy changes for that of the alkylthiol. These observations suggest that the alcohol inhibits L-1 by binding of the hydrophobic alkyl tail to the catalytic site of the enzyme by a hydrophobic interaction. The alkylthiol inhibits by binding of the nucleophilic sulfhydryl head to a polarizable region of the enzyme and the alkyl tail to a hydrophobic region of the enzyme free from the steric hindrance as an anchor.     

The minimal structural requirement of concanavalin A that retains its functional aspects

A systematic investigation of the effects of several commonly used detergents on the conformation and function of concanavalin A at pH 7 in solution form was made by using circular dichroism (CD), intrinsic fluorescence, 1-anilino 8-sulphonic acid (ANS) binding, dynamic light scattering (DLS) and sugar inhibition assay. In the presence of 6.0 mM sodium dodecyl sulphate (SDS), an anionic detergent, and 0.8 mM cetyl tri methyl ammonium bromide (CTAB), a cationic detergent, intermediate states of concanavalin A were obtained having a negative CD peaks at 222 and 208 nm respectively, a characteristic of alpha-helix. These states also retained tertiary contacts with altered tryptophan environment and high ANS binding (exposed hydrophobic area) which can be characterized as molten globule states. Concanavalin A in the presence of 5.0 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulphonate (CHAPS), a zwitterionic detergent, and 0.07 mM brij-35, a non-ionic detergent, also exists in intermediate states. These intermediates (molten globules) had high ANS binding with native-like secondary (inherent beta-sheet) and tertiary structure. The intermediate states were characterized further by means of dynamic light-scattering measurements and kinetic data. To study the possible functional requirement of the minimum structure, the intermediate states characterized in the presence of detergents were shown to retain the activity with polysaccharide (dextran). The pattern of activity observed was brij-35 > CHAPS > CTAB > SDS. The specific binding and activity of concanavalin A with ovalbumin was investigated as a function of time by turbidity measurements. Cationic and anionic detergents showed significant effects on the structure of concanavalin A as compared with zwitterionic and non-ionic detergents.     

Structure-activity analysis of the activation of pertussis toxin

Bordetella pertussis, the causative agent of whooping cough, releases pertussis toxin in an inactive form. The toxin consists of an A protomer containing one S1 peptide subunit and a B oligomer containing several other peptide subunits. The toxin binds to cells via the B oligomer, and the S1 subunit is activated and expresses ADP-ribosyltransferase and NAD glycohydrolase activities. Treatment of purified toxin with dithiothreitol (DTT) in vitro increases both activities. ATP and the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) synergistically reduce the A0.5 (activation constant) for DTT from greater than 100 mM to 200 microM. We studied the structure-activity relationships of activators of the toxin. In the presence of CHAPS (1%) and DTT (10 mM) the following compounds increased the NAD glycohydrolase activity of the toxin with the following A0.5's in microM and fraction of the ATP effect in parentheses: ATP, 0.2 (1.0); ADP, 6 (0.8); UTP, 15 (0.7); GTP, 35 (0.6); pyrophosphate, 45 (0.7); triphosphate, 60 (0.6); tetraphosphate, greater than or equal to 170 (greater than or equal to 0.4). Thus, the polyphosphate moiety is sufficient to stimulate the toxin, and the adenosine moiety confers upon ATP its extraordinary affinity for the toxin. Phospholipid and detergents could substitute for CHAPS in the activation of the toxin. Glutathione substituted for DTT with an A0.5 of 2 mM, a concentration within the range found in eucaryotic cells. Thus, membrane lipids and cellular concentrations of glutathione and ATP are sufficient to activate pertussis toxin without the need for a eucaryotic enzymatic process.     

Removal of nonionic detergent from proteins fractionated by electrofocusing.

The nonionic detergent Nonipol TD 12 (an alkyl polyoxyethylene alcohol) has been removed from solubilised proteins after their fractionation by electrofocusing. Following electrofocusing in nonionic detergent an anionic or cationic detergent was added to the focussing medium and the focusing was allowed to continue. The ionic detergent formed mixed micelles with the nonionic detergent. Thus charged, the mixed micelles migrated to either electrode, removing nonionic detergent from the fractionated proteins. Applying this technique to an adipose tissue preparation, detergent-inhibited activity of a lipolytic enzyme was restored and the binding of adenosine 3':5'-cyclic monophosphate (cyclic AMP) to a protein kinase was increased.     

Kinetics of insulin binding and kinase activity of the partially purified insulin receptor from human skeletal muscle

The kinetics of insulin binding and kinase activity of soluble, partially purified insulin receptors from human skeletal muscle are considered. An equilibrium for insulin binding was obtained within 2 h at 37 degrees C. At lower temperatures the equilibrium for insulin binding was less clearly defined. Dissociation of 125I-labelled insulin was incomplete unless an excess amount of unlabelled insulin was added. Insulin-stimulatable autophosphorylation of the 95 kDa subunit was verified by gel electrophoresis. The kinase activity was measured with the synthetic polypeptide poly(Glu-Tyr(4:1] as a phosphoacceptor. The insulin receptor kinase activity correlated significantly (r = 0.92, P less than 0.0001) to the concentration of high-affinity insulin binding sites in the eluate. Autophosphorylation of the insulin receptor was necessary for the activation of the receptor kinase. When activated the receptor kinase activity was stable for at least 60 min at 21 degrees C with a pH optimum of approx. 7.8, similar to the pH optimum for insulin binding. The non-ionic detergent Triton X-100 inhibited the sensitivity of the receptor kinase to insulin. Insulin stimulated the Vmax of the kinase reaction about 3-fold, decreased the Km for ATP from 35 +/- 5 microM (mean +/- S.E.) to 8 +/- 1 microM (P less than 0.02) and induced a positive cooperativity to ATP with an increase in the Hill coefficient from 1.00 +/- 0.02 to 1.37 +/- 0.07 (P less than 0.05). According to the Hill plots, insulin itself showed no cooperativity with respect to receptor binding or kinase activation.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

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)     

Regeneration of bacteriorhodopsin in mixed micelles

Regeneration of bacteriorhodopsin from bacterioopsin and all-trans-retinal was studied in a mixed micelle system consisting of dodecyl sulfate, CHAPS and a water-soluble phospholipid dihexanoylphosphatidylcholine (hex2-PhosChol). Regeneration to approximately 40,000 M-1.cm-1 extinction at 550 nm (epsilon 550) was obtained with either 2.3 mM or 6.5 mM CHAPS along with 6.9 mM dodecyl sulfate and 4.5 mM hex2-PhosChol in 0.16 M NaCl and 40 mM phosphate (pH 6.0). Without CHAPS, the regeneration in 4.5 mM Hex2-PhosChol gave epsilon 555 = 27,800; without PhosChol, the 1:3 CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 20,000; and without PhosChol the nearly equimolar CHAPS/dodecyl sulfate mixture gave epsilon 550 approximately 10,000. The composition of the mixed micelles was estimated from fluorescence spectroscopy using pyrene butyryl hydrazine. The molecular weight was estimated by molecular seive chromatography to be 87,100 for 2.3 mM CHAPS, 6.9 mM dodecyl sulfate and 0.67 mM hex2-PhosChol; and 83,200 for 7.0 mM CHAPS, 6.9 mM dodecyl sulfate, and 1.1 mM hex2-PhosChol. These results are consistent with the idea that at low concentrations of CHAPS and dodecyl sulfate, CHAPS organizes the dodecyl sulfate into disk shaped bilayer micelles that are favorable for bacterioopsin refolding. However, a high concentration of either detergent inhibits regeneration. Added hex2-PhosChol can overcome the inhibitory effects of high concentrations of either CHAPS or dodecyl sulfate.     

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.