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.     

The size and detergent binding of membrane proteins.

Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.     

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.     

Quantitative determination by derivative spectrophotometry of Triton X-100 in solubilized preparations of membrane proteins

The amount of the nonionic detergent Triton X-100 in protein preparations cannot be determined spectrophotometrically from the normal spectrum because the spectral bands of the detergent and proteins overlap, but it was found that the detergent could be determined accurately by 4th-derivative spectrophotometry. The intensity of the derivative absorbance difference between the positive and negative peaks at about 277.5 and 281 nm, respectively, increased linearly with increase in the detergent concentration at more than its critical micelle concentration (about 0.03%). Proteins had little effect on these spectral bands of the detergent. This method is very simple and accurate for determination of the concentration of Triton X-100 in solubilized preparations of membrane proteins.     

Estimation of labile sulfide in iron-sulfur proteins.

Lowry's method (1) for protein determination is subject to interference from the nonionic detergent Triton X-100 (2,3) which is used in high concentrations (1–5%) to solubilize membrane proteins or enzymes (4–6) and structural acidic proteins (7). Hartree (3) could reduce the errors caused by 0.1% Triton X-100 by a modification of Lowry's method. However, when protein solutions containing 0.2% or more of the detergent are mixed with the Folin-Ciocalteu reagent (1) a precipitate forms that interferes with the assay. We could reduce this interference to an insignificant level either by centrifuging the precipitate and incorporating Triton X-100 in both the reagent blank and standards, or by removing the detergent prior to the assay. This report presents two simple procedures for the Lowry assay of dilute protein samples containing 1–5% Triton X-100.     

Limited proteolysis of the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000. The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100. Removal of Triton X-100 results in association of tetramers to form octamers.     

Spectrophotometric method for quantitative determination of nonionic, ionic and zwitterionic detergents

Detergents serve as means of solubilizing biological membranes and thus play an important role in purification and characterization of membrane proteins. We report here a simple method to estimate the amount of detergent bound to a protein or present in an aqueous solution. The method is based on the turbidity caused by the addition of a detergent to triolein. Detergent bound to an integral membrane protein, lysophosphatidic acid acyltransferase, was separated by native gel electrophoresis and the amount of detergent bound to the same was estimated. This method is applicable for Triton X-100, sodium dodecyl sulfate and zwitterionic detergent, and was validated in the presence of reagents commonly used in membrane protein solubilization and purification.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.     

Hydrodynamic properties of the Triton X-100-solubilized chloroplast phosphate translocator

The molecular weight of the phosphate translocator isolated from spinach envelope membranes was measured in the nonionic detergent Triton X-100. The Stoke's radius of the protein-detergent complex was estimated by gel filtration. The partial specific volume was estimated by equilibrium centrifugation and by differential sedimentation in sucrose gradients containing H₂O and ²H₂O and the sedimentation coefficient was estimated from the same centrifugation experiments. The phosphate translocator-Triton X-100 complex has an apparent molecular weight of 177 500. Its high partial specific volume (0.86 cm³/g) suggests that bound detergent contributes significantly to the mass. Correcting for the bound detergent (1.9 g/g protein), a molecular weight of 61 000 for the protein moiety of the complex was calculated. These results suggest that the isolated phosphate translocator exists as a dimer. The shape of the dimer is described as a prolate ellipsoid of revolution with semiaxes calculated to be 6.59 and 1.59 nm in length.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.     

Preparative elution of proteins blotted to Immobilon membranes

Conditions for the preparative elution of proteins from Immobilon membranes after transfer of proteins to this matrix from sodium dodecyl sulfate-polyacrylamide gels have been established. Proteins were completely eluted from the membrane at room temperature by short incubation in 50 mM Tris-HCl, pH 9.0, containing 2% SDS and 1% Triton X-100. Good protein recoveries were also obtained in the same buffer containing 1% Triton X-100 only. The efficiency of elution was practically independent of the molecular weight of proteins, the method allowed for the precise excision of protein bands, and the proteins eluted from the matrix were not degraded. In some cases it was possible to recover enzymatic activity of the eluted proteins.     

Purification of an Acidic Structural Protein from rat Skin Using Triton X-100

An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid.     

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.     

Purification of an acidic structural protein from rat skin using Triton X-100.

An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid.     

Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Histone gene expression in early development of Xenopus laevis

Abstract. This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis , and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3.
Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.