Binding of adenovirus and its external proteins to Triton X-114. Dependence on pH

35S-Labeled adenovirus type 2 (Ad2) (10 ng/ml) was incubated with 1% Triton X-114 at various pH values varying from 3.0 to 8.0. The detergent phase was separated from the aqueous phase by centrifugation, and the amounts of Ad2 were determined in the two phases. At pH 7.0-8.0, less than 5% of Ad2 was associated with the detergent phase; at pH 5.0 or below, about 60% of Ad2 was associated with the detergent phase. When a mixture of 35S-labeled capsid proteins was used at pH 7.0, 60-70% of the total proteins were associated with the detergent at pH 5.0, but less than 5% of the proteins interacted with detergent at pH 7.0. Among the three major external proteins (hexon, penton base, and fiber), penton base had the highest association with Triton X-114 at pH 5.0. Both intact virus and the capsid proteins that were associated with Triton X-114 at pH 5.0 were released into the aqueous phase on subsequent incubation at pH 7.0. On the basis of these results, it is suggested that mildly acidic pH induces amphiphilic properties in adenovirus capsid proteins and may help Ad2 escape from acidic endocytic vesicles.     

A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.     

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.     

温度诱导双水相体系分离芦荟多糖的分配行为研究

本文研究了芦荟多糖在温度诱导双水相体系中的分配行为,考察了Triton-114的浓度、温度、酸度、盐的浓度等因素对芦荟多糖分配行为的影响。结果表明,芦荟多糖趋于分配在水相,当Triton-114浓度为4%,pH=3,温度50℃时,芦荟多糖在水相中的回收率达到最大,多糖的含量也由原来的68.39%增加到75.63%。实验还表明,无机盐对芦荟多糖的分配行为具有很大的影响。     

Biphasic interaction of Triton detergents with the erythrocyte membrane.

Octylphenoxy polyoxyethylene ethers (Triton detergents) interact with the erythrocyte membrane in a biphasic manner, i.e. they stabilize erythrocytes against hypo-osmotic haemolysis at low concentrations (0.0001-0.01%, v/v), but become haemolytic at higher concentrations. This biphasic behaviour was demonstrated with Triton X-114, Triton X-100 and Triton X-102. However, a critical chain length is a prerequisite for the haemolytic effect, because Triton X-45, which differs from the other Tritons only by the shorter chain of the polyoxyethylene residue, does not exhibit this biphasic behaviour, but goes on protecting against osmotic rupture up to saturating concentrations. Even a 1% solution of Triton X-45 does not cause haemolysis. This structural specificity of Triton X-45, namely the lack of haemolysis and efficient stabilization against osmolysis even at higher concentrations of the detergent, is exhibited at 0 degree and 37 degrees C as well as at room temperature. Three conclusions are reached: (i) a critical chain length of the octylphenoxy polyoxyethylene ethers is required for the haemolytic effect; (ii) the different structural requirements would suggest that different mechanisms are responsible for the haemolytic and the stabilizing effect of amphiphilic substances; (iii) the results suggest that haemolysis is not caused simply by dissolution of the membrane by the detergent but is a rather more specific process.     

Double Triton X-114 Phase Partitioning for the Purification of Plant Cytochromes P450 and Removal of Green Pigments

A double Triton X-114 phase partitioning procedure that separates plant cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase partitioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partitionings were carried out using phosphate buffer. We found that the partitioning of the cytochromes P450 could be shifted to a pigment-free Triton X-114 poor phase by changing the buffer component to borate. The protein extract containing the cytochromes P450 but devoid of green pigment was subjected to a second phase partitioning step before which the buffer was changed from borate to phosphate. This second phase partitioning step produced a Triton X-114-rich phase highly enriched in cytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages of purification using column chromatography. The double phase partitioning procedure takes 3–4 h to complete. This is very fast compared to traditional purification schemes for cytochromes P450 which involve multiple of column chromatographic steps. Plant cytochromes P450 are labile, low abundant proteins that are difficult to isolate. The double Triton X-114 phase partitioning here reported thus constitutes a versatile, efficient purification procedure circumventing many of the problems previously encountered.     

Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

A beta-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85 degrees C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100 degrees C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90 degrees C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k(cat)/K(m) values high enough for hydrolysis of beta-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of beta-linked glucose dimer more hydrophilic than aryl- or alkyl-beta-D-Glcp.     

A pH-induced increase in hydrophobicity as a possible step in the penetration of colicin E3 through bacterial membranes

Exposure to low pH triggers an increase in the hydrophobicity of the colicin E3 molecule. Using a [3H] Triton X-100 binding assay we have shown that the amount of detergent (at supramicellar concentrations) associated with colicin E3 increased dramatically at pH 3.8 and below. Interaction of colicin E3 with asolectin vesicles was monitored by following its cross-linking with two different photoactivatable radioactive phospholipid analogues. At neutral pH colicin E3 was cross-linked with the phospholipid probing the membrane surface whereas at pH 4.5 and below, the bacteriocin reacted with the phospholipid probing the hydrophobic core of the bilayer. With the use of phase partitioning of proteins in Triton X-114 it was shown that at acidic pH whole colicin E3 and its immunity protein segregated in the detergent phase. After trypsin digestion of the colicin-immunity complex, the N-terminal portion of E3 (T1) and the immunity partitioned in the detergent phase at low pH. In contrast, the enzymic domain of the colicin (T2) remained in the aqueous phase and was recovered in a highly active form as a consequence of its dissociation from the immunity protein. These results are discussed in relation to the mechanism of entry of colicin E3 into bacterial cells.     

Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by Triton X-100 for efficient production of d-malate

Pseudomonas pseudoalcaligenes can only form d-malate from maleate after incubation of the cells with a solvent or a detergent. The effect of the detergent Triton X-100 on d-malate production was studied in more detail. The longer the cells were incubated with Triton X-100, the higher was the d-malate production activity, until the maximal malease activity was reached. Incubation of P. pseudoalcaligenes cells with Triton X-100 also resulted in an increase in the protein concentration of the supernatant, indicating that cell lysis had occurred. The rate at which the d-malate production activity increased was dependent on the Triton X-100 concentration and on the cell density. Also the rate at which lysis occurred depended on the Triton X-100 concentration.     

TOXIN EXTRACTION FROM THE BLUE-GREEN ALGA MICROCYSTIS AERUGINOSA BY DIFFERENT EXTRACTION MEDIA

SUMMARY

The extractability of toxin from Microcystis aeruginosa isolate UV-006 by different extraction media such as O, 1N HC1, 2N CH₃COOH, Triton X-100 and water at different pH levels was investigated. The best recovery of toxin was achieved with water at a pH of 10 while the recoveries with the more acidic media tended to be lower.     

Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114.

The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 micron to 0.1-0.15 micron. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 degrees C were divided into aqueous and hydrophobic phases after incubation at 37 degrees C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organisms. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.     

Phase separation temperatures of mixtures of Triton X-114 and Triton X-45: application to protein separation.

Triton X-114 solutions separate above 22 degrees C into two immiscible aqueous phases. The more dense phase is enriched in detergent, and the less dense phase is depleted of detergent, relative to the original single phase. This phenomenon has been used to partition proteins according to hydrophobicity. The phase separation temperature is sensitive to the length of the polyoxyethylene headgroup. When Triton X-45, with a shorter headgroup, is mixed with Triton X-114 in various proportions, the phase transition temperature can be adjusted anywhere between 0 and 22 degrees C. Partitioning properties of the resulting mixtures are similar to those of Triton X-114 alone.     

Effects of temperature on the autolytic enzyme system of Streptococcus faecalis.

The cellular autolytic reaction system in Streptococcus faecalis ATCC 9790 was analyzed for relative increases in reaction rates with increasing temperature by determination of Arrhenius activation energies (E). The systems examined were: (i) an isolated wall-enzyme complex in 0.01 M sodium phosphate, pH 6.9; (ii) exponential-phase cells suspended in 0.01 or o.3 M sodium phosphate pH 6.8, or in 0.04 M ammonium acetate, pH 6.8, (iii) growing cultures deprived of glucose or lysine; and (iv) cultures treated in growth media with the nonionic detergent, Triton X-100. For detergent-treated cells, E values were between 23.9 and 27.4 kcal/mol (ca. 100.1 to 174.7 kJ/mol) at concentrations of Triton X-100 between about 0.03 and 0.072 mg/ml. E values dropped sharply to 11.5 to 13.0 kcal/m-l (ca. 48.2 to 54.4 kJ/mol) at Triton X-100 concentrations of 0.12 mg/ml or higher. For the remaining systems, E values ranged from 16 to 20 kcal/mol (ca. 67.0 to 83.7 kJ/mol) (wall lysis, cellular autolysis in 0.01 M sodium phosphate or in 0.04 M ammonium acetate, and autolysis of glucose-starved cells) to 31 to 38 kcal/mol (ca 129.8 to 159.1 kJ/mol) (cellular autolysis in 0.3 M sodium phosphate or autolysis of lysine-starved cells). High concentrations of Triton X-100 appear to lower the E values below the 16 to 20 kcal/mol observed for the autolysis of isolated walls. This effect may be related to disruption by the detergent of a hydrophobic complex regulating cellular autolysis in vivo.     

An apparatus to control and automate the formation of continuous density gradients

Triton X-114 temperature transition extraction has been considered to be a simple and cost-effective strategy to eliminate endotoxin from plasmid preparations. However, a repeated cooling-heating process may promote the degradation of plasmid DNA. Based on the finding that the cloud point of Triton X-114 solution increases substantially in the presence of small amounts of sodium dodecyl sulfate (SDS) and that electrolytes decrease the cloud point of Triton X-114-SDS solution drastically, we designed a Triton X-114 isothermal extraction method for removing endotoxin from plasmid samples and found that it has the same endotoxin removal efficiency when compared with the temperature transition extraction method.     

Studies on the hydrophobic properties of sphingomyelinase.

Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.     

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.     

Phase separation of integral membrane proteins in Triton X-114 solution

A solution of the nonionic detergent Triton X-114 is homogeneous at 0 degrees C but separates in an aqueous phase and a detergent phase above 20 degrees C. The extent of this detergent phase separation increases with the temperature and is sensitive to the presence of other surfactants. The partition of proteins during phase separation in solutions of Triton X-114 is investigated. Hydrophilic proteins are found exclusively in the aqueous phase, and integral membrane proteins with an amphiphilic nature are recovered in the detergent phase. Triton X-114 is used to solubilize membranes and whole cells, and the soluble material is submitted to phase separation. Integral membrane proteins can thus be separated from hydrophilic proteins and identified as such in crude membrane or cellular detergent extracts.     

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.     

Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10)

Carboxypeptidase E (CPE), a peptide hormone-processing enzyme, is present within secretory granules in both a soluble form and a form which is membrane-bound at pH 5.5 but soluble at neutral pH. Antisera raised against a peptide corresponding to the predicted COOH-terminus of CPE bind to the membrane-associated form of CPE but not to the soluble form. This COOH-terminal region is predicted to form an amphiphilic alpha-helix, containing several pairs of hydrophobic residues separated by hydrophilic residues. Synthetic COOH-terminal peptides 11-24 residues in length are able to bind to bovine pituitary membranes and can be extracted by conditions that extract the membrane-bound form of CPE. The influence of pH on the membrane binding of a 21-residue COOH-terminal peptide is similar to the membrane binding of CPE: at pH values less than 6 the majority of the peptide is membrane-bound, while at pH values above 8 less than 20% is membrane-bound. Both the 21-residue COOH-terminal peptide and the purified membrane form of CPE, but not the soluble form, partition into Triton X-114 only at low pH (pH less than 6). Combined polar and hydrophobic interactions of the COOH-terminal peptide appear to be responsible for the reversible, pH-dependent association of CPE with membranes.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.