Integral membrane protein interaction with Triton cytoskeletons of erythrocytes

The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.     

Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule

Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.     

Monitoring of detergent binding to amphiphilic proteins by means fo micelles containing the hydrophobic dye Sudan Black B

A simple method for detecting micellar binding of Triton X-100 to amphiphilic proteins is described. The hydrophobic dye Sudan Black B is incorporated into Triton micelles. Binding of the coloured micelles to serum apoliproteins, as well as to amphiphilic proteins, of erythrocyte and fat globule membranes renders these visible as dark bands after sucrose density gradient centrifugation. In contrast, the hydrophilic proteins present in lipoprotein-free serum do not show detergent binding. The method does not permit accurate quantification of detergent binding, but may serve as a pilot procedure for initial detection of amphiphilic proteins and for monitoring their isolation from crude solubilized membrane material. The sensitivity of the assay corresponds to that obtained with [³H]Triton X-100.     

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.     

Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).     

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of d-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards d-glucose compared to other sugars tested was shown as well as the main features of d-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of d-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the d-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 Å; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65 000. We conclude that this protein fraction is involved in d-glucose transport by renal brush borders.     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Endocytosis of GPI-linked membrane folate receptor-alpha

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36- 38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100- resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.     

Adaptation of the bicinchoninic acid protein assay for use with microtiter plates and sucrose gradient fractions

The bincinchoninic acid protein assay has been adapted for use with microtiter plates and a plate reader to reduce the time needed for analyses. The total volume of the assay was reduced to 0.21 ml with various ratios of sample to reagent volume being used. The assay is sensitive to a limit of less than 1 microgram of bovine serum albumin. The assay is insensitive to the presence of the detergents sodium dodecyl sulfate, Triton X-100, and deoxycholate. In addition, the method has been adapted for determining the protein concentrations of sucrose density gradient fractions, which eliminates the need for removing the sucrose prior to assay.     

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.     

PROPERTIES OF MEMBRANE-BOUND HEXOKINASE IN RAT BRAIN

Abstract— —-(1) The total hexokinase activity present in the mitochondrial fraction can be solubilized completely by incubation with salt and Triton X-100. This activity cannot be entirely released by washing with sucrose or by freezing and thawing.
(2) A part of the particle bound hexokinase exists in a latent form. The latent form is apparent after incubation with high salt concentrations, detergents or by freezing and thawing.
(3) Solubilization of membrane bound hexokinase is pH-dependent. Incubation in salt solutions increases the specific activity ten-fold. The salt concentration and pH are con-current. At pH 7.0 part of the hexokinase is solubilized. The lower the pH the less salt is required to release the same amount of activity.
(4) Triton X-100 solubilizes particle-bound hexokinase, but to a less extent than salts. The activation of hexokinase is greater with Triton X-100 than with salt.
(5) The possible nature of the bonds between hexokinase and mitochondrial membranes is discussed.     

c-Abl proto-oncoprotein is expressed and tyrosine phosphorylated in human sperm cell

The presence and possible role of c-Abl proto-oncoprotein was investigated in human sperm cell. The c-Abl monoclonal antibody (mAb), against the protein tyrosine kinase domain of v-Abl protein, reacted specifically with the acrosomal region of methanol-fixed capacitated and non-capacitated human sperm cell in the indirect immunofluorescence technique. The c-Abl mAb predominantly recognized two protein bands of 145 kD and 95 kD in detergent-solubilized (Triton X-100 and NP-40) sperm and testes preparations in the Western blot procedure. The 95 kD protein band reacted stronger than the 145 kD band and was the only band detected in the lithium diiodosalicylate (LIS)–solubilized sperm preparation, and even in the Triton X-100/NP-40 extracts of sperm of some men. In the in vitro kinase assay using the Triton X-100–solubilized capacitated sperm preparation, the 95 kD protein was autophosphorylated at the tyrosine residues, which was inhibited in the presence of c-Abl mAb. The tyrosine phosphorylation of sperm proteins, especially of the 95 kD protein, has been shown to have a vital role in human sperm function, namely, the sperm capacitation/acrosomal exocytosis and binding to zona pellucida of oocyte. These findings suggest that the c-Abl or c-Abl-like proteins are present in mature sperm cells that are tyrosine autophosphorylated and may have a role in human sperm cell function. Mol. Reprod. Dev. 51:210–217, 1998. © 1998 Wiley-Liss, Inc.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

On-column purification and refolding of recombinant bovine prion protein: using its octarepeat sequences as a natural affinity tag

Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases. Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies. Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni-NTA agarose column. Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations. Triton X-100 and beta-mercaptoethanol were required in this rapid refolding process. The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy. Its correct folding was confirmed from circular dichroism (CD) experiments. Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP(sc).     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Plasma-membrane-associated immunoglobulins and other polypeptides of pig mesenteric node lymphocytes.

1. Xanthine oxidase (EC 1.2.3.2) was found to represent more than 8% of the intrinsic protein of the bovine milk-fat-globule membranes. 2. Less than 25% of the xanthine oxidase activity of the fat-globule membrane was solubilized with 0.1 M-sodium pyrophosphate buffer or 2M-NaCl. Of the particulate activity remaining 56% was solubilized with Triton X-100. 3. The xanthine oxidase activity solubilized with buffer, 2M-NaCl or Triton X-100 was not liberated as the free enzyme. Only tryptic digestion was found to release the free enzyme from the fat-globule membrane. Tryptic digestion also liberated free xanthine oxidase from those fractions solubilized by buffer or NaCl, but not from those fractions solubilized with Triton X-100 or by sonication. 4. The effect of membrane association on the catalytic properties of the enzyme could be mimicked by low pH or by the presence in the assay mixture of certain concentrations of 2-methyl-propan-2-ol, but not 1,4-dioxan, suggesting that hydrogen-bonding rather than low dielectric constant may be involved. 5. The origin of the milk-fat-globule membrane is discussed with reference to the intrinsic nature of the associated xanthine oxidase activity.     

Proportional equilibration of K, Na ions, and sucrose molecules in pig lenses incubated in the presence of the non-ionic detergent Triton X-100

The release of sodium and potassium and the uptake of sucrose molecules was studied in pig lenses incubated in isosmotic sucrose solution in either the presence or absence of 1% Triton X-100 (a non-ionic detergent). This Triton X-100 treatment has been shown to cause severe disruptions of cell membrane integrity. If sodium and potassium were free in the lens fibers as in a dilute aqueous solution, they would be expected to diffuse three to four times faster than sucrose. However, measurements of sodium and potassium release and sucrose uptake in the Triton X-100 treated lenses show a 1:1 equilibration. When pig lenses were incubated in the same solution without detergent, the sucrose uptake was significantly less than the potassium and sodium release. It is postulated that a slow, detergent mediated collapse of protein-water-ion interactions within the lens is the rate-limiting step of the observed equilibration of monovalent cations and sucrose molecules.     

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.     

Virus-receptor interaction in the adenovirus system I. Identification of virion attachment proteins of the HeLa cell plasma membrane.

Plasma membranes from HeLa cells were isolated in a two-phase polymer system. To compare the efficiency of attachment protein extraction, a normalized assay for the assessment of adenovirus type 2 (Ad2) receptor-active components interfering with the attachment of Ad2 to HeLa cells was developed. An optimized detergent extraction procedure, 0.5% Triton X-100, was used, and solubilized membrane proteins were radioisotope labeled in vitro. Proteins with affinity for Ad2 virions were quantified and identified in a sucrose gradient sedimentation assay and by affinity chromatography with cross-linked Ad2 virions immobilized to AH-Sepharose 4B. From virions recovered in the sucrose gradient system, one major membrane component of high affinity was identified with a polypeptide molecular weight of around 40,000. Glycosylated proteins isolated by wheat germ lectin chromatography with high affinity for immobilized virus particles were isolated, and two major components with apparent molecular weights of 40,000 and 42,000 were identified. We suggest that a glycosylated protein with high affinity for Ad2 virions and a polypeptide molecular weight of 40,000 to 42,000 is one component of the Ad2 attachment site on HeLa cells.