Two-phase Partitioning Method Research Articles

Coupling with in-situ electrochemical reactive chlorine species generation and two-phase partitioning method for enhanced microalgal biodiesel production

Microalgal-based biofuel production is of great significance in alleviating energy crisis and achieving carbon neutrality. However, the excessive costs and high solvent consumption in lipids extraction from microalgal obstruct the widespread application of biodiesel in practice. Reported herein is the construction of facile strategy for lipids extraction via electrocatalytic pretreatment and a subsequent two-phase partitioning method. Electrocatalytic pretreatment method adopts the solar as power source and avoids the drying of microalgal biomass, in favor of carbon neutrality requirement. During this process, eco-friendly electrode with high specific surface area could contribute to the sufficient generation of reactive chlorine species (RCS), facilitating the outflows of intracellular lipid. As a result, assisted with two-phase partitioning method, a satisfied performance of lipid recovery (86.72 %) was obtained. Notably, compared with traditional solvent method, two-phase partitioning method greatly reduced the dosage of organic solvent, which is an economical or environmental technique.

A Protocol for the Plasma Membrane Proteome Analysis of Rice Leaves.

Subcellular proteome analysis is one of the most effective ways to reduce the complexity of total proteome. With the advancement in protein extraction methodologies, it is now possible to fractionate and isolate the proteins from subcellular compartments without significant contamination from the cytoplasm and other organelles. Of the different subcellular proteomes, plasma membrane remained largely uncharacterized because of the difficulties in isolation of contamination free plasma membrane proteins. Moreover, proteome analysis in the past two decades majorly relied on the two-dimensional gel electrophoresis which showed limited protein loading ability and poor separation of highly hydrophobic plasma membrane proteins. Development of shotgun proteomics methods has facilitated the identification and quantification of hydrophobic proteins isolated from plasma membrane or other cellular membranes. Here, we present a simplified procedure for the isolation of plasma membrane proteins by a two-phase partitioning method and their identification by shotgun proteomics approach using rice as a model plant.

Choline priming-induced plasma membrane lipid alterations contributed to improved wheat salt tolerance

Salt stress is a major environmental threat influencing crop growth and yield. The plasma membrane (PM) is believed to be one facet of the cellular mechanisms of salt adaptation. Choline priming has been reported to enhance salt tolerance of the sensitive wheat cultivar used in this work. The study was, therefore, undertaken to examine whether changes in the PM lipids will participate in choline-improved salt tolerance. The caryopses were primed in choline chloride (0, 5 and 10 mM) for 24 h. They were then germinated in sand for 10 days, watered with 1/4-strength modified Hoagland solution (MHS). The seedlings were grown in the sand, watered with MHS containing 150 mM NaCl for 3 weeks. Root PM was isolated by two-phase partitioning method and its lipid classes were determined. Choline maintained the PM total lipids, sterol and phospholipids, which were altered by NaCl. The PM sterols/phospholipids ratio was decreased by NaCl, whereas choline retained this ratio. Salt stress reduced the PM unsaturated fatty acids while increased its saturated fatty acids. Choline alleviated PM unsaturated/saturated ratio reduction. NaCl declined the PM phosphatidylcholine (PC), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG) whereas increased phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Choline decreased PS and PE, while increased PC level. The PM cholesterol, campesterol and β-sitosterol were increased while stigmasterol was declined under NaCl. Choline increased stigmasterol whereas decreased cholesterol and campesterol. The alterations in the PM lipids were discussed in relation to choline-enhanced salt tolerance.

Characterization of Ca2+/H+ exchange in the plasma membrane of Saccharomyces cerevisiae

The characteristics of the Ca2+/H+ exchange were directly investigated in functionally inverted (inside-out) plasma membrane vesicles isolated from yeast using an aqueous two-phase partitioning method. Results showed that following the generation of an inside-acid pH gradient (fluorescence quenching), addition of Ca2+ caused movement of H+ out of the vesicles (fluorescence recovery). The Ca2+/H+ exchange displayed saturation kinetics with respect to extravesicular Ca2+ and ATP concentrations in the plasma membrane, and showed specificity for Ca2+. The protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), abolished the fluorescence quenching and consequently inhibited Ca2+/H+ exchange in plasma membrane vesicles. Vanadate, which is known to inhibit the plasma membrane H+-ATPase, significantly decreased the Ca2+-dependent transport of H+ out of vesicles. When the electrical potential across the plasma membrane was dissipated with valinomycin and potassium, the rate of Ca2+/H+ exchange increased compared to that of the control without valinomycin, indicating that the stoichiometric ratio for this exchange is greater than 2H+:Ca2+. These data suggest that Ca2+ is transported out of yeast cells through a Ca2+/H+ exchange system that is driven by the proton-motive force generated by the plasma membrane H+-ATPase.

Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca2+/H+ exchangers have been identified in endomembranes, but far fewer have been studied for Ca2+/H+ exchange in plasma membrane so far. To investigate the Ca2+/H+ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca2+/H+ exchange in plasma membrane vesicles was measured by Ca2+-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca2+/H+ exchange was optimal at pH 7.0, and displayed transport specificity for Ca2+ with saturation kinetics at Km = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca2+-dependent transport of H+ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca2+/H+ exchange increased comparing to control without valinomycin effect, suggesting that the Ca2+/H+ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H+:Ca2+. Eosin Y, a Ca2+-ATPase inhibitor, drastically inhibited Ca2+/H+ exchange in plasma membrane as it does for the purified Ca2+-ATPase in proteoliposomes, indicating that measured Ca2+/H+ exchange activity is mainly due to a plasma membrane Ca2+ pump. These suggest that calcium (Ca2+) is transported out of Arabidopsis cells mainly through a Ca2+-ATPase-mediated Ca2+/H+ exchange system that is driven by the proton-motive force from the plasma membrane H+-ATPase.

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel-based and a LC MS/MS-based proteomics method. Two-day-old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two-phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel-based proteomics, four and eight protein spots were identified as up- and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up- and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low-abundance proteins could be identified by the LC MS/MS-based method. Three homologues of plasma membrane H(+)-ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H(+)-ATPase protein.

Characterization of Amyloid β Fibrils with An Aqueous Two-Phase System: Implications of Fibril Formation

The pathological process of Alzheimer’s disease is closely related to amyloid fibril formation by the causative protein, amyloid  (A). The growth behavior of A fibrils is predominated by the seeds-monomeric A interaction. In this study, the local hydrophobicity of seeds of A fibrils was investigated by the aqueous two-phase partitioning method to evaluate the hydrophobic interaction between the seed-monomeric A. The seeds showed a high local hydrophobicity relative to the monomer and fibrils. From the fibril growth experiment and the additive effect of Triton X-100, we could demonstrate the contribution of the hydrophobic seeds-monomer interaction to fibril formation.

Analysis of Plasma Membrane Proteome in Soybean and Application to Flooding Stress Response

The plasma membrane acts as the primary interface between the cellular cytoplasm and the extracellular environment. To investigate the function of the plasma membrane in response to flooding stress, plasma membrane was purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method. Purified plasma membrane proteins with 81% purity were analyzed using either two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry and protein sequencing (2-DE MS/sequencer)-based proteomics or nanoliquid chromatography followed by mass spectrometry (nanoLC-MS/MS)-based proteomics. The number of hydrophobic proteins identified by nanoLC-MS/MS-based proteomics was compared with those identified by 2-DE MS/sequencer-based proteomics. These techniques were applied to identify the proteins in soybean that are responsive to flooding stress. Results indicate insights of plasma membrane into the response of soybean to flooding stress: (i) the proteins located in the cell wall are up-regulated in plasma membrane; (ii) the proteins related to antioxidative system play a crucial role in protecting cells from oxidative damage; (iii) the heat shock cognate protein plays a role in protecting proteins from denaturation and degradation during flooding stress; and (iv) the signaling related proteins might regulate ion homeostasis.

Proteome Analysis of Rice Root Plasma Membrane and Detection of Cold Stress Responsive Proteins

To investigate the function of plant plasma membrane, proteins of rice plasma membrane were analyzed and the proteins changed by cold stress were identified. Plasma membrane proteins were purified with an aqueous two-phase partitioning method from root of rice seedlings, and activity of specific H(+)-ATPase localized in plasma membranes was measured. The plasma membrane proteins were separated by SDS-PAGE or 2D-PAGE, and analyzed with nano LC-MS/MS. The number of transmembrane helices was predicted from the amino acid sequence of annotated proteins. Functional categorization revealed that the most of proteins were associated with energy production, signal transduction, protein synthesis, cell growth/division and defense. In addition, 12 cold stress responsive proteins were identified from the plasma membrane using 2D-PAGE based proteomics method. Out of them, cold shock protein-1 was significantly decreased in plasma membrane of rice under cold stress.

Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation

It has previously been suggested that plasma membrane ATPase (PM H+-ATPase, EC 3.6.1.3.) is a site of incipient freezing injury because activity increases following cold acclimation and there are published data indicating that activity of PM H+-ATPase is modulated by changes in lipids associated with the enzyme. To test and extend these findings in a tree species, we analyzed PM H+-ATPase activity and the fatty acid (FA) composition of glycerolipids in purified plasma membranes (PMs) prepared by the two-phase partition method from current-year needles of adult red pine (Pinus resinosa Ait.) trees. Freezing tolerance of the needles decreased from -56 degrees C in March to -9 degrees C in May, and increased from -15 degrees C in September to -148 degrees C in January. Specific activity of vanadate-sensitive PM H+-ATPase increased more than two-fold following cold acclimation, despite a concurrent increase in protein concentration. During de-acclimation, decreases in PM H+-ATPase activity and freezing tolerance were accompanied by decreases in the proportions of oleic (18:1) and linoleic (18:2) acids and increases in the proportions of palmitic (16:0) and linolenic (18:3) acids in total glycerolipids extracted from the plasma membrane fraction. This pattern of changes in PM H+-ATPase activity and the 18:1, 18:2 and 18:3 fatty acids was reversed during cold acclimation. In the PM fractions, changes in FA unsaturation, expressed as the double bond index (1 x 18:1 + 2 x 18:2 + 3 x 18:3), were closely correlated with changes in H+-ATPase specific activity (r2 = 0.995). Changes in freezing tolerance were well correlated with DBI (r2 = 0.877) and ATPase specific activity (r2 = 0.833) in the PM fraction. Total ATPase activity in microsomal fractions also closely followed changes in freezing tolerance (r2 = 0.969). We conclude that, as in herbaceous plants, simultaneous seasonal changes in PM H+-ATPase activity and fatty acid composition occur during cold acclimation and de-acclimation in an extremely winter hardy tree species under natural conditions, lending support to the hypothesis that FA-regulated PM H+-ATPase activity is involved in the cellular response underlying cold acclimation and de-acclimation.

Integration of a Two-Phase Partition Method into Proteomics Research on Rat Liver Plasma Membrane Proteins

To comprehensively identify proteins of the rat liver plasma membrane (PM), we have adopted a proteomics strategy that utilizes sucrose density centrifugation in conjunction with aqueous two-phase partition for plasma membrane isolation, followed by SDS-PAGE, mass spectrometry and bioinformatics. Western blot analysis showed that this method results in highly purified plasma membrane fractions, which is a key to successful plasma membrane proteomics. The PM proteins were separated by SDS-PAGE and digested with trypsin. Through nano-ESI-LC MS/MS analysis we identified 428 rat liver membrane proteins, of which 304 had a gene ontology (GO) annotation indicating a cellular component, and 204 (67%) of the latter were known integral membrane proteins or membrane-associated proteins. In addition to proteins known to be associated with the plasma membrane, several hypothetical proteins have also been identified. This study not only provides a tool to study plasma membrane proteins with low levels of contamination, but also provides a data set for proteins of high to moderate abundance in rat liver plasma membranes, thus allowing for more comprehensive characterization of membrane proteins and a better understanding of membrane dynamics.

Biological consequences of petite mutations in Candida glabrata

To define the pathogenicity of respiration-deficient mutants of Candida glabrata, which present a reduced susceptibility to azoles, that are easily induced in vitro by exposure to these drugs or to ethidium bromide and that may be selected in vivo in patients receiving fluconazole. Two wild-type isolates of C. glabrata were compared with their respective fluconazole- or ethidium bromide-induced petite mutants, regarding the carbohydrate and protein composition of the cell wall, as well as their surface physical properties, and also their adherence abilities and virulence in mice. Flow cytometric analysis of cell wall carbohydrates using several fluorescent lectins showed an increased binding of mutant cells to concanavalin A compared with their parent isolates, suggesting a greater availability or an increased amount of glucose-mannose residues at the cell surface in petite mutants. Likewise, some quantitative differences between parent and mutant isolates were shown by SDS-PAGE in protein extracts from blastoconidia. Regarding the surface physical properties, no significant differences were seen in the electrophoretic mobility determined by microelectrophoresis, but the two-phase partitioning method revealed a lower cell surface hydrophobicity for petite mutants. Moreover, mutant cells exhibited significant overexpression of CgEPA1 as revealed by real-time reverse transcription-PCR, but the adherence capacities to Caco-2 cells, a human enterocyte line, were not significantly different. Finally, in agreement with their slower growth, petite mutants were less virulent than parent isolates in a murine model of systemic infection. This low virulence in mice suggests that petite mutants could be disregarded clinically although they may arise during fluconazole therapy.

Two-phase partition chromatography using soybean oil eliminates pesticide residues in aqueous ginseng extract

This study was carried out to develop a cost-, labor- and efficiency-effective elimination method of pesticide residues in ginseng extract. A two-phase partition method between soybean oil and distilled water or aqueous ginseng extract was employed for the elimination of pesticide residues. Content of the pesticides was determined by gas chromatography with electron capture or nitrogen phosphorus detector. A total of 15 pesticides representing four categories (organochlorine, organophosphorus, carbamate, pyrethroid) were spiked (ca. 2 ppm) to 2 ml soybean oil in a test tube and the oil was mixed with 6 ml distilled water or 10% aqueous ginseng extract. The test tubes were then vortexed (2 min) and centrifuged at 3000 rpm for 15 min to separate the oil and aqueous layers. Each layer was harvested and subjected to quantitative analysis of pesticides. The average distribution ratio of the pesticides to the oil layer was 94.4±6.7% in the mixture of the oil and distilled water, and 105.5±6.6% in the mixture of the oil and ginseng extract. No significant qualitative and quantitative change of ginsenosides, the active ingredients of Panax ginseng, was observed in the ginseng extract before and after the oil treatment. These results suggest that two-phase partition chromatography between soybean oil and the aqueous phase is a cost-, labor- and efficiency-effective reliable method for the elimination of pesticide residues in ginseng extract.

Permeability behaviour of lipid vesicles prepared from plant plasma membranes—impact of compositional changes

Exposure of oat seedlings to repeated moderate water deficit stress causes a drought acclimation of the seedlings. This acclimation is associated with changes in the lipid composition of the plasma membrane of root cells. Here, plasma membranes from root cells of acclimated and control plants were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracted from the plasma membranes. In a series of tests the vesicle permeability for glucose and for protons were analysed and compared with the permeability of model vesicles. Further, the importance of critical components for the permeability properties was analysed by modifying the lipid composition of the vesicles from acclimated and from control plants. The purpose was to add specific lipids to vesicles from acclimated plants to mimic the composition of the vesicles from control plants and vice versa. The plasma membrane lipid vesicles from acclimated plants had a significantly increased permeability for glucose and decreased permeability for protons as compared to control vesicles. The results point to the importance of the ratio phosphatidylcholine (PC)/phosphatidylethanolamine (PE), the levels of cerebrosides and free sterols and the possible interaction of these components for the plasma membrane as a permeability barrier.

Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition. PMs from root cells of plants grown in excess copper (50 μM Cu 2+) and control (0.3 μM Cu 2+) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress. Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.

Enhanced ATP-dependent copper efflux across the root cell plasma membrane in copper-tolerant Silene vulgaris.

We studied copper uptake in inside-out plasma membrane vesicles derived from roots of copper-sensitive, moderately copper-tolerant and highly copper-tolerant populations of Silene vulgaris (Amsterdam, Marsberg and Imsbach, respectively). Plasma membrane vesicles were isolated using the two-phase partitioning method and copper efflux was measured using direct filtration experiments. Vesicles derived from Imsbach plants accumulated two and three times more copper than those derived from Marsberg and Amsterdam plants, respectively. This accumulation was ATP-dependent. Also, 9-amino-6-chloro-2-methoxyacridine fluorescence quenching rates upon copper addition decreased in the order Imsbach>Marsberg>Amsterdam. Our results support the hypothesis that efflux of copper across the root plasma membrane plays a role in the copper tolerance mechanism in S. vulgaris.

Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning.

Effective extraction of protein from bulk medium is an important technique in bioresearch. In the present study, we describe an extracellular beta-xylosidase from the fermentation supernatant of Trichoderma koningii G-39 that was successfully extracted and purified simultaneously in a single step by using an aqueous two-phase partitioning method. This two-phase system was prepared by dissolving suitable amount of poly(ethylene glycol) (PEG) and sodium dihydrogenphosphate (NaH(2)PO(4)) in aqueous solution. beta-Xylosidase was recovered with high yield and high concentration in the bottom salt-rich phase when 25% (w/v) PEG 1500 and 20-25% (w/v) NaH(2)PO(4) were applied. Based on a 1-liter scale extraction, the purity of the enzyme was enhanced at least 33-fold. The total activity increased 422% in comparison with that in the untreated filtrate. The effectiveness and simplicity may make this technique potentially useful in various applications. The transxylosylation activity of the enzyme purified by this technique was also investigated.

Simulation and System Analysis of Gene Regulatory Networks Using the Two-phase Partition Method

Simulation and System Analysis of Gene Regulatory Networks Using the Two-phase Partition Method

Influence of water stress on the lipid physical state of plasma membranes from P. betuloefolia. Bqe leaves

We use the two-phase partitioning method to purify the plasma membrane vesicles as materials, and investigate the influence of water stress on the physical state of plasma. Our experiments show that, as the concentration of PEG increases (that is, as the extent of water stress increases), the fluorescent time and even fluorescent time of long-life composition of DPH decreases, the fluorescent polarized value of DPH and the fluorescent intensity of MC 540 increase. All of these imply that water stress makes plasma membranes vesicles small, the flowing and surface charge density of plasma membrane decreases. It also suggests the hydrophobicity reduction. In our further experiments, the time of long-time composition of intrinsic tryptophane decreased, and the activity of H +-ATPase reduced, which suggests the change of configuration and function of plasma membrane proteins. As a result, we thought that the change of the lipid physical state of plasma membranes is the primary response of possible awareness to water stress.

Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50–55°C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60°C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45°C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.