Comparison of the cytoskeleton fractions of rat red blood cells prepared with non-ionic detergents

The characteristics of cytoskeleton fractions prepared from rat red cell ghosts with four non-ionic detergents were studied. One percent (w/v) solutions of Triton X-100, Emulgen 911, MEGA-9 (nonanoyl-N-methylglucamide), and octylglucoside solubilized 78, 68, 80, and 92% of the ghost phospholipid, while they solubilized 82, 78, 72, and 62% of the ghost band 3, a transmembrane protein, respectively. There was no correlation between the solubilization percentages of phospholipid and band 3. Phospholipids retained in cytoskeleton fractions were shown to exist as blebs on the surface by electron microscopic observation. The cytoskeleton fraction prepared with octylglucoside retained about two-fold more band 3 than that with Triton X-100 (Triton shells). However, cytoskeleton fractions prepared from p-chloromercuribenzoate-treated ghosts with the two detergents retained almost equal amounts of band 3, less than 5% of that in the ghosts. Under this condition, most of band 2.1, a protein linking band 3 to the spectrin-actin network, was released from the cytoskeleton fractions. The band 3 solubilized with octylglucoside sedimented faster in a linear sucrose gradient and had a larger Stokes' radius than that with Triton X-100, which is known to exist as dimer. These results strongly suggest that octylglucoside does not disturb the association of tetrameric band 3 with the spectrin-actin network, while Triton X-100 dissociates tetrameric band 3 to the dimer, resulting in the difference in the amount of band 3 retained in cytoskeleton fractions. In conclusion, octylglucoside can produce a more native cytoskeleton fraction of red cell membranes than Triton shells.     

Interaction of detergents with cytochrome c oxidase.

The binding of ionic and nonionic, nondenaturing detergents to cytochrome c oxidase has been examined. All bind and displace part but not all of the phospholipid that is associated with the enzyme after isolation. From 6 to 10 phospholipid molecules, depending on the detergent used, do not exchange and these are mostly diphosphatidylglycerol molecules as first shown by Awasthi et al. ((1971) Biochim. Biophys. Acta 226, 42). The binding of Triton X-100 and deoxycholate to the cytochrome c oxidase complex has been studied in detail. Both bind to the enzyme above their critical micelle concentrations: Triton X-100 in the amount of 180 +/- 10 molecules per complex and deoxycholate in the amount of 80 +/- 4 molecules per complex. In nonionic detergents, cytochrome c oxidase exists as a dimer (4 heme complex). The enzyme is dissociated into the monomer or heme aa3 complex by delipidation in bile salts. Activity measurements in different detergents suggest that cytochrome c oxidase requires a flexible, hydrophobic environment for maximal activity and that the dimer or 4 heme complex may be the active species.     

Properties of acid ceramidase from human spleen

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Impact of Triton X-100 on alpha 2-antiplasmin (SERPINF2) activity in solvent/detergent-treated plasma

Large-pool solvent/detergent (SD) plasma for transfusion exhibits reduced alpha 2-antiplasmin (alpha2-AP; SERPINF2) functional activity. The reason for the loss of alpha2-AP has not been described and could be due to the SD incubation itself and/or to the processing steps implemented to remove the solvent and the detergent. We have studied alpha2-AP activity during six down-scale preparations of plasma virally-inactivated by 1% (v/v) TnBP combined with two different non-ionic detergents, either 1% Triton X-100 or 1% Triton X-45, at 31 degrees C for 4h. The SD-treated plasmas were then extracted with 7.5% (v/v) soybean oil, centrifuged at 3800 x g for 30 min, and subjected to hydrophobic interaction chromatography (HIC) to remove the SD agents. Control runs without TnBP and Triton were performed to evidence possible impacts of each process step on alpha2-AP activity. TnBP, Triton X-100, and Triton X-45 were measured at all stages of the processes to evaluate potential interferences with the alpha2-AP assay. Alpha 2-AP activity was about 10% that of starting plasma after 1% TnBP-1% Triton X-100 incubation and about 50% after oil extractions, centrifugation, and HIC. By contrast about 73% of the antiplasmin activity was found after the incubation with 1% TnBP and 1% Triton X-45, 88% after removal of the SD agents by oil extractions, 90% after centrifugation and 92% after HIC. The control runs performed without SD agents showed that the process steps did not affect the alpha2-AP activity. In conclusion, the agent altering alpha2-AP activity in SD-plasma is Triton X-100. The choice of detergents for the SD viral inactivation of therapeutic plasma fractions used in patients at risk of fibrinolysis should consider the impact on alpha2-AP activity.     

Isoelectric focusing analysis of detergent extracted renal 11 beta-hydroxysteroid dehydrogenase

11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) from rat renal cortex microsomes was solubilized using several detergents, the most effective being Zwittergent 3-10 and Triton X-100. The activity ratio oxidation/reduction of the reversible reaction corticosterone in equilibrium 11-dehydrocoticosterone varied depending on the detergent used. We attribute this variation to direct effects of different detergents on enzyme kinetics. In contrast, comparable results obtained with liver 11-HSD have been attributed to the possibility of spatially separated 11-oxidase and 11-reductase activities. In order to test whether renal 11-HSD represents a uniform oxido-reductase as generally assumed, or a dual enzyme system as has been recently proposed an attempt was made to characterize 11-HSD solubilized from renal microsomal fractions using isoelectric focusing (IEF). When 11-HSD was extracted with 1% Triton X-100 (= partially solubilized fraction) a heterogenous peak pattern was obtained. In contrast, IEF of 11-HSD extracted with 10% Triton X-100 (= delipidated fraction) resulted in a single peak at about pH 5.9 with both oxidative and reductive activity at practically identical positions within the gels. From this observation we conclude that the degree of detergent solubilization of a membrane bound protein affects its amphoteric properties and that removal of membranous lipids is a prerequisite for the analysis of its behaviour. Since the more delipidated fraction of 11-HSD revealed only one activity peak the data are compatible with the uniform enzyme concept since oxidative and reductive activities of renal cortical 11-HSD could not be separated.     

Interactions between sarcoplasmic reticulum calcium adenosintriphosphatase and nonionic detergents

The interaction of Triton X-100 and other nonionic detergents with a delipidated preparation of the Ca2+ ATPase from sarcoplasmic reticulum has been studied. Binding of radiolabeled Triton X-100 was determined by column chromatography at 6 degrees C, and two classes of binding sites were observed. Below the critical micelle concentration (cmc), binding of Triton occurred at 35-40 equivalent sites on the delipidated ATPase with a binding constant of 2.7 X 10(4) M-1. Near the cmc cooperative binding of an additional 70 molecules of the detergent was observed. The binding of monomeric Triton X-100 below the cmc was associated with a parallel activation of over half of the ATPase activity, and the remainder of the activity was recovered after the detergent concentration was increased to the cmc. The ability to reactivate ATPase activity was more dependent on the polar poly(oxyethylene) portion of nonionic detergents than on the hydrocarbon portion. Generalizing for all amphiphiles, these results suggest that there are discrete binding sites on the Ca2+ ATPase for phospholipid molecules in the native membrane and that the polar head groups of phospholipids interact more strongly with the protein than the hydrophobic acyl chains. Perturbations in micelle structure were observed for several nonionic detergents by measurement of cis-parinaric acid fluorescence and differential scanning calorimetry, and discontinuities in Arrhenius plots occurred at the transition temperature of the detergent used for reactivation of ATPase activity. It is concluded that both the physiol state of teh micelle and the intrinsic behavior of the ATPase polypeptide affect the temperature dependence of ATPase activity.     

Effect of triton X-100 on the activity and solubilization of rat liver mitochondrial phosphatidylserine decarboxylase

It was shown that, among ionic and nonionic detergents tested, only Triton X-100 was able to stimulate the activity of rat liver phosphatidylserine decarboxylase, whereas other detergents were without effect or were inhibitory. The solubilization procedure of phosphatidylserine decarboxylase from mitochondrial membranes with Triton X-100 was elaborated. The dependence of the solubilized decarboxylase on the Triton X-100 to phosphatidylserine ratio and the inhibitory effect of Triton X-100 at its molar ratio to phospholipid higher than 5.6 was observed. No divalent cation requirement and no dependence of the ionic strength for the solubilized enzyme were observed. Kinetic parameters were determined.     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Solubilization of thyroid peroxidase by nonionic detergents.

We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.     

Resistance of Human Erythrocyte Membranes to Triton X-100 and C<Subscript>12</Subscript>E<Subscript>8</Subscript>

Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4°C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C₁₂E₈. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C₁₂E₈, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C₁₂E₈ in comparison to intact cells, while the difference in the S values between Triton X-100 and C₁₂E₈ DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C₁₂E₈ detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone.

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH3 pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent:protein ratios from 0.5 to 20 led to a progressive loss of hormone . receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone . receptor complex was not retained by 0.22 micron filters and remained soluble after ultracentrifugation. Following incubation with high (2.5--10%) concentrations of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent:protein ratio of 0.033. The hormone . receptor complex was included in Sepharose 6B and exhibited an apparent Stoke radius of 46 A in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0 degrees C, while the membrane hormone . receptor complex was stable for up to 5 at 0 degrees C.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH₃ pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C.     

Factors effecting the solubilisation of stearoyl-coA desaturase of hen liver microsomes.

1. The lipid requirement for maximum desaturase activity was investigated using acetone/water mixtures. It was shown that for maximum stearoyl-CoA desaturase activity of hen liver microsomes neither the total neutral lipid fraction nor 44% of the phospholipid fraction were required. 2. The effect of sodium deoxycholate, Triton X-100, Nonidet P-40 and Bio-solv on the enzyme activity indicated that the neutral detergents had a milder effect than the ionic detergent but both classes could cause considerable irreversible loss of activity. 3. The treatment of the microsomes with 2.5% (v/v) water in acetone greatly improved the effective solubilising power of Triton X-100. The yield of desaturase in the 100 000 X g supernatant obtained by treating the microsomal fraction in this way was strongly dependent upon protein concentration. Maximum solubilisation was achieved with25 mg protein per ml 1% (w/v) Triton X-100 in 0.1 M potassium phosphate buffer pH 7.4. 4. A comparison of the properties of the solubilised and membrane-bound enzyme was made by an investigation of: (i) the temperature and pH optimum, (ii) activation energy and (iii) the effect of inhibitors on the enzyme activity.     

Electrophoretically monodisperse cytochrome c oxidases

A discontinuous gradient polyacrylamide gel electrophoresis under nondenaturing conditions has been used to demonstrate monodispersity of procaryotic and eucaryotic cytochrome c oxidase preparations. Alkaline treated bovine enzyme which contains nine subunits as analysed by subsequent discontinuous SDS-polyacrylamide gel electrophoresis is a monodisperse dimer in 0.1% Triton X-100 and a monomer in 0.1% dodecyl maltoside. The Mr-values corrected for bound detergent are 286,000 in Triton X-100 and 152,000 in dodecyl maltoside respectively. The two-subunit bacterial cytochrome c oxidase of Paracoccus denitrificans is proved to be a monomer with a corrected Mr of 76,000 in both nonionic detergents Triton X-100 and dodecyl maltoside.     

Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.     

The solubilization and morphological change of human platelets in various detergents

The solubilization of human gel-filtered platelets by octyl glucoside, Triton X-100, dodecylsulfate, and deoxycholate was compared from the analysis of (1) cell lysis, (2) marker leakiness, and (3) component solubility. These analyses all revealed that the effect of detergent concentration on the solubilization of platelets by these detergents was exerted in three stages, i.e., the prelytic, lytic, and complete platelet-lysis stages. These analyses also indicated several differences among platelets in these detergents. (i) The ratio of the platelet-saturation concentration (PSC) to critical micellar concentration (CMC) was about 1/2 for octyl glucoside. Triton X-100 and dodecylsulfate, while it was close to 1 for deoxycholate. (ii) Platelets in octyl glucoside. Triton X-100, and dodecylsulfate all showed parallel curves in cell lysis, protein solubilization and marker leakiness, while the platelet lysis in deoxycholate was identical to the phospholipid solubilization. (iii) The solubility curves of various components in Triton X-100 and deoxycholate were parallel. However, the solubility of cholesterol in octyl glucoside was lower than that of protein and phospholipid. In dodecylsulfate, the solubility of phospholipid and cholesterol was very low in comparison with that of protein. In addition, morphological studies using scanning electron microscopy (scanning EM) revealed that the solubilization by octyl glucoside or Triton X-100 might occur via membrane area expansion. On the other hand, the solubilization by dodecylsulfate or deoxycholate showed membrane vesiculation prior to cell lysis. Moreover, in the prelytic stage, the morphological change in platelets in octyl glucoside showed only concentration dependence by swelling to an ellipsoid and then to a sphere. However, the morphological change in platelets in the other three detergents was dependent not only on the detergent concentration but also on prolonged incubation. Specifically, in Triton X-100, the cells initially changed to spiculate discs and then reached their final shape as swollen discs with surface invagination. In dodecylsulfate and deoxycholate the morphological changes were almost the same. The cell initially deformed in shape to a spiculate disc and finally to a stretched-out flat form. The results are discussed according to the bilayer couple hypothesis. Also, in the prelytic stage, these detergents caused inhibition of the response of platelets to collagen and ADP-fibrinogen.     

Binding, activation, and solubilization of the Ca2+-ATPase from sarcoplasmic reticulum by nonionic detergents

Interactions between delipidated Ca2+-ATPase from sarcoplasmic reticulum and four nonionic detergents--dodecyl octaoxyethyleneglycol monoether (C12E8), Triton X-100, Brij 58, and Brij 35--were characterized with respect to activation of ATPase activity, binding, and solubilization. C12E8 and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C12E8. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C12E8 and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C12E8 are more useful when bulk solubilization is the goal.     

Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Dimeric nature of apo-low density lipoprotein extracted with Triton X-100.

Human low density lipoprotein (LDL) was dissolved in 0.3 to 2.0% Triton X-100 at pH 7.5 and apo-LDL (B protein) was extracted from LDL to form B protein-Triton complex. Sedimentation equilibrium study of this complex in a solvent nearly isopycnic to Triton X-100 showed that the molecular weight of the protein in the complex was 570,000. The complex eluted almost at the void volume of a Sepharose 6B column, as would be expected for a complex with a total molecular weight of roughly 900,000, on the assumption that 0.52 g of Triton was bound to 1 g of protein (Helenius, A. and Simons, K. (1972) J. Biol. Chem. 247, 3656-3661). The sedimentation coefficient of the complex gave f/fmin = 2.2, indicating that the complex was either as asymmetric as a fibrinogen molecule or not compact. These results show that B protein exists in its complex with Triton X-100 as an elongated or a loosely expanded dimer based on the molecular weight of monomeric B protein of 270,000. B protein may also exist in LDL as a dimer.