以Triton X-114液相分离法去除质粒溶液中的内毒素

目的：采用TritonX-114液相分离法去除质粒溶液中的内毒素,以保证实验动物的安全和结果的准确性。方法：通过碱裂解法提取质粒pVAX1和pVAX1-hLDHC,用聚乙二醇6000沉淀对质粒进一步纯化;用TritonX-114抽提的方法,去除质粒溶液中的内毒素。结果：通过3轮TritonX-114抽提,能够将质粒溶液中内毒素水平降至1．95EU／mL,质粒样品的回收率为79．8％,质量保持不变。结论：TritonX-114液相分离法是一种非常有效的去除质粒溶液中内毒素的方法。     

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.     

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.     

Phase separation of rat intestinal brush border membrane proteins using Triton X-114

Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4 degrees C. Upon phase separation at 32 degrees C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and Ca2+-Mg2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization.     

Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114

Above its critical micelle concentration, Triton X-114 in solution forms two phases at room temperature: a lower phase containing supramicellar aggregates and an upper phase largely depleted of detergent. This property of the detergent is potentially useful for separating under mild conditions proteins that bind detergent from those that do not (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). We studied the distribution of the receptor for immunoglobulin E (IgE) and its subunits in the two phases. IgE and IgE complexed either with intact receptors or with the alpha chains of the receptor alone are principally partitioned into the upper phase, whereas the unliganded receptor as well as the isolated alpha, and especially the beta and gamma chains of the receptor, preferentially partition into the lower detergent phase. Chromatography of IgE and of the subunits of the receptor on a hydrophobic support showed that the beta and gamma chains have a considerably greater hydrophobic surface than the alpha chains or IgE. These results indicate that the distribution of a protein in the two phases of phase-separated Triton X-114 is not an all-or-none effect based upon whether it binds detergent or not. Rather, it reflects the overall balance between the hydrophobic and hydrophilic properties of the protein's surface.     

Two membrane-bound forms of tyrosylprotein sulfotransferase as revealed by phase partitioning in Triton X-114.

Tyrosylprotein sulfotransferase (TPST) is a membrane-associated enzyme of the trans Golgi network that catalyzes the posttranslational sulfation of a variety of secretory and membrane proteins. We have analyzed the membrane association of TPST in Golgi-enriched fractions from bovine adrenal medulla using carbonate treatment (pH 11) and Triton X-114 phase partitioning. TPST was not extracted by carbonate. Triton X-114 phase partitioning revealed that, unexpectedly, TPST from non-carbonate-treated membranes was present in both, a hydrophilic and a hydrophobic form with apparent sedimentation coefficients of approximately 13 and approximately 6, respectively. Extraction of membranes with carbonate converted the hydrophilic form TPST to the hydrophobic form. Addition of the carbonate extract to TPST solubilized from carbonate-treated membranes converted the hydrophobic form of the enzyme to the hydrophilic form. This conversion of TPST was specific in that it was not observed for the bulk of the proteins present in the carbonate-treated membranes. The factor in the carbonate extract responsible for this conversion, referred to as "phase-transfer factor", (i) was precipitable with ammonium sulfate and polyethylene glycol, (ii) was non-dialyzable, (iii) was not extracted from membranes by 0.5 M NaCl, and (iv) appeared to be more abundant than TPST itself. These results show that TPST is an integral membrane protein and suggested that the enzyme may exist in a complex with a peripheral membrane protein. Moreover, a phase-transfer factor was also observed in another system, PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach

The photosystem (PS) II antenna system comprises several biochemically and spectroscopically distinct complexes, including light-harvesting complex II (LHCII), chlorophyll-protein complex (CP) 29, CP26, and CP24. LHCII, the most abundant of these, is both structurally and functionally diverse. The photosynthetic apparatus is laterally segregated within the thylakoid membrane into PSI-rich and PSII-rich domains, and the distribution of antenna complexes between these domains has implications for antenna function. We report a detailed analysis of the differences in the polypeptide composition of LHCII, CP29, and CP26 complexes associated with grana and stroma thylakoid fractions from spinach (Spinacia oleracea L.), making use of a very high-resolution denaturing gel system, coupled with immunoblots using monospecific antibodies to identify specific antenna components. We first show that the polypeptide composition of the PSII antenna system is more complex than previously thought. We resolved at least five type I LHCII apoproteins and two to three type II LHCII apoproteins. We also resolved at least two apoproteins each for CP29 and CP26. In state 1-adapted grana and stroma thylakoid membranes, the spectrum of LHCII apoproteins is surprisingly similar. However, in addition to overall quantitative differences, we saw subtle but reproducible qualitative differences in the spectrum of LHCII apoproteins in grana and stroma membrane domains, including two forms of the major type II apoprotein. The implications of these findings for models of PSII antenna function in spinach are discussed.     

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.     

Analysis of Toxoplasma gondii proteins after Triton X-114 solubilization and hydrophobic chromatography

The distribution of the surface proteins of Toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Two polypeptides with 15,000 and 70,000 distributed in both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities. Two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of T. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.     

Ultrastructure of the Thylakoid Membrane in Tomato Leaf Chloroplast Revealed by Liquid Helium Rapid-Freezing and Substitution-Fixation Method

The ultrastructure of the chloroplast in tomato leaves was studiedby a liquid helium rapid-freezing and substitution-fixationmethod. Distinct morphological differences were observed betweenthe ultrastructure of the thylakoid membrane fixed by this methodand the one fixed by the conventional chemical fixation method.The membrane was asymmetrical and consisted of triple-layeredleaflets. While the leaflet facing the stroma (outer leaflet)was very electron-dense, the one facing the thylakoid lumen(inner leaflet) was less electron-dense. The triple-layeredleaflets showed a structural difference between the grana thylakoidand the stroma thylakoid. In the grana region, where thylakoidmembranes are appressed, the part of outer leaflets were lookedlike fused or adhered to each other making one highly electron-denselayer composed of linings of particles (about 4 nm in diameter).The inner leaflet of the grana thylakoid membrane showed discontinuousstructure. This discontinuous structure was composed of a clusterof irregular particles (about 2–3 nm in diameter). Thesize of cluster varied from about 10nm to 16nm. The discontinuityof the inner leaflet, i.e. linings of the clusters at ratherconstant intervals, was not observed clearly in the stroma thylakoid.The liquid helium rapid-freezing and substitution-fixation methodcan be used to observe the details of protein complexes in thethylakoid membrane organization. (Received March 23, 1988; Accepted August 17, 1988)     

Hydrophobicity of protochlorophyllide oxidoreductase, characterized by means of Triton X-114 partitioning of isolated etioplast membrane fractions

The inner etioplast membrane possesses a pronounced lateral heterogeneity with respect to protein and lipid composition as well as ultrastructural appearance. Little is known about the reason for formation of the regular branched structure shown by the prolamellar body part of the membrane. A specific interaction between the membrane lipids and the dominating protein NADPH-protochlorophyllide oxidoreductase (PCR, EC 1.6.99.1) might be of major importance. In this study isolated prolamellar bodies and prothylakoids from the leaves of dark-grown wheat ( Triticum aestivum L. cv. Starke II, Weibull) were exposed to Triton X-114 partitioning in media with 150 m M NaCl and without. By comparing the partitioning of PCR, the ATP synthase (EC 3.6.1.3) polypeptides, and ribulosebisphosphate carboxylaseoxygenase (EC 4.1.1.39) in the different systems, it was concluded that PCR is an integral membrane protein with substantial hydrophilic domains.     

Phase separation of Triton X-100 micelle solution induced by osmotic stress

We have found out that the phase separation of Triton X-100 micelle solution was caused by the addition of poly(ethylene glycol) (PEG) above a critical concentration. The critical concentration of PEG depended on its molecular weight and temperature, larger molecular weight or higher temperature giving lower critical concentration. These results were analyzed on the basis of the osmoelastic coupling theory recently proposed by us (Biochemistry (1989) 28, 3710-3715; Biochemistry (1989) 28, 5626-5630).     

Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114

The detergent Triton X-114, because of its convenient cloud point temperature (22 °C), has been used extensively to extract membrane proteins and to separate them in two phases according to their hydropathy. The upper detergent-poor phase contains mostly hydrophilic proteins, whereas hydrophobic ones are found mainly in the lower detergent-rich phase. In this work, we developed a method to fractionate membrane proteins and estimate their hydropathy based on a series of cloud point partitions with Triton X-114. With this method, beetroot plasma membrane proteins were separated in different fractions according to their hydropathy, following the binomial distribution law as expected. This method revealed the presence of both hydrophilic and hydrophobic Ca²⁺-dependent protein kinases in those membranes. At least five distinct Ca²⁺-dependent kinases were observed in in-gel kinase activity assays. This separation procedure was also used as the first step in the purification of a hydrophobic 60-kDa kinase.     

Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114.

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatii]) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.     

Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N-glycosylation

BackgroundNumerous proteins depend on correct glycosylation for their proper function and nearly all membrane, as well as secreted, proteins are glycosylated. Glycosylation of membrane proteins plays a crucial role in many processes including the intercellular recognition and intermolecular interactions on the cell surface. The composition of N-glycans attached to membrane proteins has not been sufficiently studied due to the lack of efficient and reproducible analytical methods.MethodsThe aim of this study was to optimise cloud-point extraction (CPE) of membrane proteins with the non-ionic detergent Triton X-114 and analyse their N-glycosylation using hydrophilic interaction liquid chromatography (HILIC-UPLC). Purification of isolated proteins from the excess of detergent proved to be the key step. Therefore, several purification procedures were tested to efficiently remove detergent, while retaining maximum protein recoveries.ResultsCPE showed to be an efficient method to simultaneously extract membrane and soluble proteins, which subsequently resulted in different N-glycan profiles of the aforementioned protein groups. The resulting protocol showed satisfactory reproducibility and potential for N-glycan analysis of both membrane and intracellular (soluble) proteins from different kinds of biological material.ConclusionsThis method can be used as a new analytical tool for reliable detection and quantification of oligomannose and complex type N-glycans attached to membrane proteins, thus serving to distinguish between differences in cell types and states.General significanceThe simple method was successfully optimised to generate reliable HILIC-UPLC profiles of N-glycans released from membrane proteins. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

Two Polypeptides in the Inner Chloroplast Envelope of Dunaliella tertiolecta Induced by Low CO(2)

Unicellular green algae have a mechanism for concentrating dissolved inorganic carbon (DIC) only when grown in low CO₂. To find proposed transporter protein(s) for DIC, we isolated intact chloroplasts from Dunaliella tertiolecta cells, separated the chloroplast envelopes by isopyknic centrifugation, and separated their polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two peptides of apparent molecular masses of 45 and 47 kD were constituents of the inner chloroplast envelope only if the cells had been adapted to low CO₂ in the light or grown in low CO₂. These two low CO₂-induced peptides appear to be part of the algal DIC pump.     

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.     

Demonstration of the amphiphilic character of hormone-sensitive lipase by temperature-induced phase separation in Triton X-114 and charge-shift electrophoresis

Temperature-induced phase separation in Triton X-114 (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607) and charge-shift electrophoresis (Helenius, A., and Simons, K. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 529-532) were used to examine the amphiphilic character of hormone-sensitive lipase, purified from rat adipose tissue. In contrast to ATP-citrate lyase, a reference hydrophilic protein, the lipase was shown to partition predominantly (approximately 80%) into the detergent-rich phase upon phase separation in Triton X-114. Furthermore, its electrophoretic mobility was markedly shifted anodally and cathodally upon charge-shift electrophoresis in the presence of sodium taurodeoxycholate and cetyltrimethylammonium bromide, respectively. The results demonstrate that hormone-sensitive lipase possesses detergent-binding hydrophobic domain(s) and exhibits the same amphiphilicity as typical intrinsic membrane proteins.