Aqueous Dextran-polyethylene Glycol Two-phase System Research Articles

Characterization of low temperature-induced plasma membrane lipidome remodeling combined with gene expression analysis reveals mechanisms that regulate membrane lipid desaturation in Carica papaya

Carica papaya is a cold-sensitive horticultural crop, the mechanisms underlying its cold sensitivity remains unclear which limit its genetic improvements in cold tolerance. In this study, aqueous dextran-polyethylene glycol two-phase partition systems were applied to purify plasma membrane from papaya leaves grown under 28 °C and 10 °C, the plasma membrane total lipids were analyzed by lipidomic approach; meanwhile, the transcription levels of putative fatty acid desaturases were quantified by RT-qPCR. We found that cerebrosides are dominate plasma membrane lipid species, followed by phospholipids and free sterols. Cold treatment increased the mono-unsaturated and di-unsaturated phospholipids, and decreased di-saturated phospholipids, the proportion of total phospholipids was not affected. Cold stress enhanced the transcription of CpFAD4, CpFAD6 and CpFAB2−2. Pathway analysis suggested that the cold induction of CpFAB2−2 transcription and CpFAD2 enzyme activities play important roles for plasma membrane lipidome remodeling under cold stress. Our findings uncovered potential targets to improve papaya cold tolerance.

Structural features important for differences in protein partitioning in aqueous dextran–polyethylene glycol two-phase systems of different ionic compositions

Partitioning of 15 proteins in dextran–70-polyethylene glycol (PEG)-8000 aqueous two-phase systems (ATPSs) in the presence of 0.01M sodium phosphate buffer, pH7.4 was studied. The effect of salt additives (NaCl, CsCl, Na2SO4, NaClO4 and NaSCN) at different concentrations on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the protein partition coefficients in ATPSs with and without salt additives. The results obtained show that the presence and concentration of salt additives affect the protein partition behavior. Analysis of ATPSs in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning could not be explained by the protein size. The structural signatures for the proteins were constructed from partition coefficient values in four ATPSs with different salt additives, and the structural distances were calculated using cytochrome c as the reference structure. The structural distances for all the examined proteins (except lysozyme) were found to be interrelated. Analysis of about 50 different descriptors of the protein structures revealed that the partition behavior of proteins is determined by the peculiarities of their surfaces (e.g., the number of water-filled cavities and the averaged hydrophobicity of the surface residues) and by the intrinsic flexibility of the protein structure measured in terms of the B-factor (or temperature factor).

Membrane Preparation, Sucrose Density Gradients and Two-phase Separation Fractionation from Five-day-old Arabidopsis seedlings

Membrane preparation has been widely used for characterization the membrane proteins. Membrane fractions can be separated by a combination of differential and density-gradient centrifugation techniques (Hodges et al., 1972; Leonard and Vanderwoude, 1976). Here we firstly describe a method to isolate total microsomal fractions including plasma membrane, intracellular vesicles, Golgi membranes, endoplasma reticulum, and tonoplast (vacuolar membrane) from 5-7 days old seedlings, which is often analyzed for auxin transporters in Arabidopsis (Leonard and Vanderwoude, 1976; Titapiwatanakun, et al., 2009; Yang et al., 2013; Blakeslee et al., 2007). After homogenization, plant debris including cell walls, chloroplasts and nucleus were removed by low speed centrifugation (8,000 x g), then total microsomal membranes were pelleted by high speed centrifugation (10,000 x g) and separated from soluble fractions. We secondly describe a method to separate microsomal fractions according to size or density in a sucrose density-gradient system by centrifugation. The linear sucrose gradient from 20%-55% (1.09-1.26 g cm-3) were used to separate membranes with different densities: tonoplast, 1.10-1.12 cm-3, Golgi membranes, 1.12-1.15 cm-3, rough endoplasmic reticulum 1.15-1.17 cm-3, thylakoids, 1.16-1.18 cm-3, plasma membrane, 1.14-1.17 g cm-3, and mitochondrial membranes, 1.18-1.20 cm-3 (Leonard and Vanderwoude, 1976; Larsson et al., 1987; Briskin and Leonard, 1980). However, the plasma membrane can also be isolated according to its outer surface properties which are very different from intracellular membrane surfaces. Thus, the right-side-out plasma membrane vesicles can be separated in an aqueous Dextran-polyethylene glycol two-phase system. The plasma membranes can be purified to > 90% in the upper phase (Larsson et al., 1987; Alexandersson et al., 2008). Two-phase systems for Arabidopsis seedlings were described in the section 3. Sucrose density gradient membrane fractionation followed by western blot is often used to analyze the distribution of certain membrane protein, while Two-phase separation is used when high purity of plasma membrane or intracellular membrane is required.

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.

Characterization of plasma membrane domains enriched in lipid metabolites.

A subpopulation of plasma membrane vesicles enriched in membrane lipid metabolites has been isolated from petals of carnation flowers and leaves of canola seedlings. This was achieved by immunopurification from a microsomal membrane preparation using region-specific antibodies raised against a recombinant polypeptide of the plasma membrane H(+)-ATPase. The properties of this subpopulation of vesicles were compared with those of purified plasma membrane isolated by partitioning in an aqueous dextran-polyethylene glycol two-phase system. The lipid composition of the immunopurified vesicles proved to be clearly distinguishable from that of phase-purified plasma membrane, indicating that they represent a unique subpopulation of plasma membrane vesicles. Specifically, the immunopurified vesicles are highly enriched in lipid metabolites, including free fatty acids, diacylglycerol, triacylglycerol and steryl and wax esters, by comparison with the phase-purified plasma membrane. These findings can be interpreted as indicating that lipid metabolites generated within the plasma membrane effectively phase-separate by moving laterally through the plane of the membrane to form discrete domains within the bilayer. It is also apparent that these domains, once formed, are released as vesicles into the cytosol, presumably by microvesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.

Do glycerolipids display lateral heterogeneity in the thylakoid membrane?

The lateral heterogeneity of lipids in the thylakoid membrane has been questioned for over 20 yrs. It is generally believed that glycerolipids are asymmetrically distributed within the plane of the membrane. In the present investigation, we isolated several thylakoid membrane domains by using sonication followed by separation in an aqueous dextran-polyethylene glycol two-phase system. This technique, which avoids detergent treatments, allowed us to obtain stroma and grana lamellae vesicles as well as grana central core and grana margin vesicles from thylakoids. The relative distribution of the four lipid classes, i.e., monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, and phosphatidylglycerol, was found to be statistically identical in all four thylakoid fractions and in whole thylakoids. Similarly, the relative amount of fatty acids in each individual lipid and the eight main phosphatidylglycerol molecular species was identical in all thylakoid membrane fractions tested as well as in the intact thylakoid membrane. Based on presently available procedures for obtaining thylakoid subfractions that are unable to discriminate microdomains within the membrane, it is concluded that glycerolipids are evenly distributed within the plane of the thylakoid membrane. These data are discussed in terms of "bulk" and "specific" lipids.

Peptides partitioning in an aqueous dextran–polyethylene glycol two-phase system

Partitioning of glycine, lysine and aspartic acid and their oligopeptides in an aqueous dextran–polyethylene glycol two-phase system containing 0.15 M NaCl in 0.01 sodium phosphate buffer, pH 7.3 and 0.11 M sodium phosphate buffer, pH 7.3 was examined. Relative hydrophobicity of the amino acid residues and peptide bonds was estimated and expressed in equivalent numbers of methylene units. Analysis of a series of reversed di- and tripeptides in terms of relative hydrophobicity showed that the additivity principle does hold for the hydrophobicity of short peptides. The relative hydrophobicity of peptides is affected by the ionic composition of aqueous media as well as by the type of amino acid residues forming peptide bonds in a given peptide sequence.

The topology of phosphatidylglycerol populations is essential for sustaining photosynthetic electron flow activities in thylakoid membranes

The transmembrane distribution of phosphatidylglycerol (PG) was determined in rightside-out (RO) and inside-out vesicles (IO) obtained by fragmentation of spinach thylakoids in a Yeda press, followed by partition in an aqueous dextran-polyethyleneglycol two-phase system. Using the phospholipase A 2 from porcine pancreas to digest selectively PG molecules in the outer monolayer (exposed to the incubation medium) of the membrane, we found the molar outside/inside distribution to be 70/30±5 in RO and 40/60±3 in IO. The transmembrane distribution of PG in IO was the opposite of that in intact thylakoids (molar ratio 58/42±3). The phospholipid population which sustained most of the uncoupled photosystem II electron flow activity was localized in the inner monolayer (exposed to the thylakoid lumen) of both thylakoid and RO membranes. In contrast, the activity in IO membranes was highly dependent on the PG population located in the outer monolayer. This finding brings the first direct demonstration of the dependence of the photosynthetic electron flow activity on the integrity of the inner topological pool of PG in the thylakoid membrane.

The transmembrane distribution of galactolipids in spinach thylakoid inside-out vesicles is opposite to that found in intact thylakoids

The transmembrane distribution of galactolipids has been determined in spinach thylakoid inside-out vesicles obtained by fragmentation in a Yeda Press followed by partition in an aqueous dextran-polyethylene glycol two-phase system. Using the lipase from Rhizopus arrhizus to digest selectively the galactolipids in the outer monolayer of the membrane, we have found the molar outside/inside distribution to be 42±1/58±1 for monogalactosyldiacylglycerol (MGDG) and 82±0.2/18±0.2 for digalactosyldiacylglycerol (DGDG). As expected, the transmembrane distribution of galactolipids in inside-out vesicles was the opposite to that found in intact thylakoids which, under similar conditions, was found to be 62/38 for MGDG and 20/80 for DGDG. The reliability of the enzymatic approach to determine the transmembrane distribution and the relative arrangement of acyl lipids in the thylakoid membrane are discussed.

Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.

An affinity-ligand gradient technique for purification of enzymes by counter-current distribution

Enzymes related to the glycolysis, present in an extract of baker's yeast, have been fractionated by counter-current distribution using an aqueous dextran—polyethyleneglycol two-phase system containing affinity ligands. To minimize the peak width a gradient of a general affinity ligand (triazine dye) was included in the stationary (liquid) phase. A centrifugal counter-current distribution machine was used to speed up the settling of the aqueous phases after equilibration. With 55 transfer cycles several of the measured enzymes increased 4–15 times in purity. Furthermore, a number of the enzymes showed clear heterogeneity indicating two or more isoenzymic forms. The use of this multistep liquid—liquid extraction in large scale for specific isolation of intracellular enzymes is discussed.

ATP-dependent activation of glucocorticoid-receptor complexes from the rat's heart

The effect of ATP on the cytosolic rat heart glucocorticoid receptor was studied by employing different methods for evaluation of the changes in molecular properties of the receptor, induced by activation. Incubation of triamcinolone acetonide labelled cytosol at 25°C or with 10 mM ATP at 4°C leads to the increase in the partition coefficient of the receptor in the aqueous dextran-polyethylene glycol two-phase system and also nuclear uptake of the complexes. The effect of ATP on the partition coefficient was more pronounced, compared with that of thermal treatment or dilution of the cytosol and totally inhibited by 10 mM sodium molybdate. The activating effect of ATP on the glucocorticoid-receptor complexes and sensitivity of this activation to sodium molybdate was also confirmed by DEAE-cellulose chromatography of cytosolic receptor preparations. The results suggest that ATP may be involved in the glucocorticoid receptor activation and through this regulates the translocation of complexes into the nucleus under in vivo conditions.

Lipoteichoic acid is the major cell wall component responsible for surface hydrophobicity of group A streptococci.

The contribution of lipoteichoic acid (LTA) to the hydrophobic surface properties of group A streptococci was investigated in aqueous dextran-polyethylene glycol two-phase systems. Enzymatic digestions were performed to characterize the hydrophobic surface structure. The results obtained indicated that LTA is a major factor responsible for the hydrophobic character of the cell surface of group A streptococci. This was further supported by the similarity of partition in polymer two-phase systems between whole group A streptococci and tritiated LTA extracted from a group A streptococcal strain. Surface LTA was also determined on intact organisms by a new method measuring the adsorption of antibodies to LTA to the bacterial surface. A correlation was found between the content of surface LTA and the hydrophobicity of the group A streptococci. We conclude that surface-associated LTA is the major factor determining surface hydrophobicity of group A streptococci.

Binding of antibiotics to the bacterial ribosome studied by aqueous two-phase partitioning

The present article describes the use of the aqueous dextran-polyethylene glycol two-phase system for the study of the interactions between the bacterial ribosomes and some antibiotics like streptomycin, chloramphenicol, tobramycin, and tetracycline. As compared to other methods, such as equilibrium dialysis, this simple technique appears to be a particularly suitable and rapid one.

Inside-out membrane vesicles isolated from spinach thylakoids

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl- p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.

Conversions of the glucocorticoid · receptor complex of rat thymocyte cytosol, studied by partition in an aqueous dextra-polyethylene glycol two-phase system

Tritiated glucocorticoid · receptor complex was formed by incubation of [ 3H] triamcinalone acetonide at –5°C with thymocyte cytosol, made 40% with respect to glycerol. The cytosol was then incubated at various temperatures, ionic strengths and cytosol concentrations for various times. At the end of the incubations, the partition coefficient of the complex in an aqueous dextranpolyethylene glycol two-phase system was determined. By the criterion of time-dependent changes of the partition coefficient, two differential conversions of the complex were distinguished: 1. A rapid, ionic strength-dependent process, increasing the partition coefficient. 2. A slower, temperature-, ionic strenght- and cytosol concentration-dependent process, increasing the partition coefficient. The effect of the salt composition of the partition mixture on the partition coefficient indicated, that the complex has a p I below 6. The effect of pH of the partition mixture indicated that the conformation of the state of aggregation of the complex is dependent on pH.

Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer. The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural characteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H 2O to phenyl- p-benzoquinone and ascorbate-Cl 2Ind to NADP + electron transport rates indicate a ratio of around six between Photosystem II and I. The total fractionation procedure could be completed within 2–3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP +. These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.

Transformation of glucocorticoid-receptor complex from rat thymocytes and its accumulation in chromatin in a cell-free system

The accumulation of glucocorticoid-receptor complex from rat thymocyte cytosol in a thymocyte chromatin preparation has been studied. A thymocyte 100 000 × g supernatant was prepared and the receptor stabilized by the addition of glycerol until 40%. Tritiated glucocorticoid-receptor complex was formed by incubation of this solution with tritiated glucocorticoids at −5°C. The chromatin accumulated part of the complex at incubations at 4°C. Receptor without hormone was not accumulated in the chromatin. The accumulation from cytosol diluted and preincubated at 4°C prior to the addition of the chromatin occurred with a high rate, whereas a low rate was seen without preincubation. This indicated a transformation of the complex during the preincubation. This transformation was found to be obligatory for the accumulation and to be promoted by dilution of the supernatant and by high ionic strength. The transformed and the untransformed complexes differed with respect to partition coefficients in an aqueous dextran-polyethylene glycol two-phase system and in their behaviour during adsorptions with dextran-coated charcoal, where great loss of transformed complex was observed. The accumulation of complex in the chromatin was found to be unsaturable in the concentration interval studied (0.07–0.25 nM).

Separation of subchloroplast membrane particles by counter-current distribution

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicates that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.

Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin

Specific cell surface membrane receptors, labeled by forming a complex with low concentrations (about 10--9 M to 10--10 M) of a highly radioactive (125-I, carrier-free) ligand, can serve as simple, reliable, sensitive, and quantitative markers for plasma membranes in fractionation procedures. 125-I-Labeled insulin, cholera toxin and the plant lictins, wheat germ agglutinin (WGA), and concanavalin A are the receptor ligands used for labeling plasma membranes. Plasma membranes are labeled before homogenization by incubating intact cells briefly at 24 degrees or 4 degrees, or by very brief in situ perfusion of the organ, with the 125-I-Labeled marker. After removing the free 125-I-labeled ligand from the medium by washing (at 4 degrees), the membrane-marker complex remains intact over prolonged (days) periods of time at 4 degrees. Labeling occurs nearly exclusively on the cell surface, the specificity of this plasma membrane reaction is maintained through homogenization and fractionation, and little dissociation of the complex, detectable exchange of label, or aggregation occur even upon prolonged incubation of the homogenates. When desired, the complex can be dissociated deliberately by manipulating experimental conditions such as temperature or by adding specific simple sugars. The most generally suitable marker appears to be WGA. At least in certain tissues (e. g. fat cells) labeling of the plasma membrane with 125-I-WGA and 125-I-isnulin can be performed equally well and selectively in homogenates as in the intact cell. 125-I-Cholera toxin cannot be used in homogenates because of significant binding to nuclei. The use of 125-I-labeled WGA as a specific plasma membrane marker is illustrated in following the course of fractionations, and in quantitating the yield and purity, of plasma membranes from fat cells, lymphocytes, and liver. The results are compared with simultaneous measurements of the plasma membrane enzyme "markers," ATPase, 5-nucleotidase, and basal as well as hormone-stimulated adenylate cyclase activities. The fractionation of liver plasma membranes by aqueous dextran-polyethylene glycol two-phase polymer systems and by conventional differential centrifugation procedures arealso quantitated with the marker, 125I-WGA. Substantial quantities of plasma membrane material are no recovered in the interphase of the two-phase polymer system. Conventional liver fractionation procedures which retain, for further purification, only the readily sedimented pellet (2000 times g, 15 min) discard a very large (at least 70%) questenal hy