Development of a general ligand for immunoaffinity partitioning in two phase aqueous polymer systems

The effect on the partition of erythrocytes in a two phase aqueous polymer system based on dextran T500 and polyethylene glycol (PEG) 8000 of a combination of immunoaffinity ligands, namely, rabbit immunoglobulin G (IgG) and PEG 1900-modified monoclonal IgG, was examined as a potential cell separation technique. Several hybridoma lines secreting mouse monoclonal IgG specific for the Fc receptor of rabbit IgG were raised. The monoclonal IgG was modified by cyanuric chloride attachment of PEG 1900, causing the modified antibody to partition predominantly into the PEG-rich upper phase of the systems. The PEG-modified monoclonal IgG was used as an affinity ligand in the two phase polymer system to specifically increase the partition of rabbit anti-NN glycophorin IgG. The rabbit IgG was applied together with the PEG-modified monoclonal IgG to increase the partition of human erythrocytes. The same system had no effect on the partition of rabbit erythrocytes. These experiments demonstrate that a monoclonal antibody can be modified and used as a general reagent with which to alter cell partition in two phase aqueous polymer systems in an immunologically specific manner.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems.

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6 degrees C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maxium occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging.The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6°C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to sialic acid-associated cell surface charges. The pattern of change in relation to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maximum occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Vancomycin partitioning in aqueous two-phase systems: effects of pH, salts, and an affinity ligand

The partitioning of vancomycin in polyethylene glycol (PEG)-dextran and PEG-phosphate aqueous two-phase systems was studied at different pHs, at varying concentrations of neutral salts, and with an affinity ligand attached to methoxy polyethylene glycol (MPEG). Vancomycin is found to partition preferentially into the PEG-rich top phase, and its partition coefficient increases nearly exponentially with the addition of water structure-making salts, such as sodium sulfate and sodium chloride, but is independent of sodium phosphate concentration. In the PEG-dextran system the vancomycin partition coefficient increases 3-fold in acidic and neutral solutions, while in the PEG-phosphate system it increases about 30-fold on the addition of the same amount of sodium chloride (1. 5 mol/kg). In basic solution, above its isoelectric point, the vancomycin partition coefficient increases slightly with NaCI concentration in the PEG-dextran system. We also examined the use of the dipeptide D-ala-D-ala as an affinity ligand on MPEG to extract vancomycin into the PEG-rich phase. The vancomycin partition coefficient increased almost 7-fold upon adding the MPEG-ligand in an amount equal to approximately 3% of the total PEG in the system. Finally, fractionation of the polydisperse phase-forming polymers in the two-phase PEG-dextran system was observed. The effect of this polymer fractionation on the partition coefficient of vancomycin is discussed.     

Possibility of analytical application of the partition in aqueous biphasic polymeric systems technique

The partition behaviour of a number of ionic and nonionic surface-active substances in the dextran-polyethylene glycol system was examined. The strictly linear dependence of the logarithm of the partition coefficient on the alkyl chain length for a homologous series of nonionic surfactants provides a measure of the difference in the relative hydrophobicity between the two phases of the system, in terms of the free energy of transfer of a CH₂ group from the bottom phase to the top phase of the system. This difference is found to be altered in the presence of NaCl or KCl depending on the salt concentration. It is concluded that the influence of the salt composition of the system on the distributed solutes' behaviour may be due to the effect of the ions on the hydrophobicity difference between the phases.The partition of ionic amphiphiles is found to be dependent on the relative hydrophobicity of the compounds as well as on their charge. It is shown that at salt concentrations up to about 0.1 M NaCl the charged solute partition is determined by its charge as well as its relative hydrophobicity, in the presence of 0.1–0.2 M NaCl the substance distribution is highly dependent on its charge and slightly on its lipophility. At the salt concentrations above 0.2 M the solute partition is determined just by its hydrophobic character and seems to be totally independent of its charge. It is concluded that the partition technique can be used for analytical purposes. The method seems to be unique in providing quantitative information on the amphiphilic surface properties of the solutes being partitioned.     

Interplay between plasmid partition and postsegregational killing systems

Active partition systems and postsegregational killing (PSK) systems are present together in naturally occurring low-copy-number plasmids. Theory suggests that PSK may act as the ultimate determinant of plasmid retention, whereas the partition system may minimize the growth penalty to the host, resulting in a near-ideal symbiosis when the systems combine. Here, we prove the validity of this principle for a specific case involving the P1par system and the mvp PSK system.     

The effect of variable liposome brightness on quantifying lipid-protein interactions using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has been increasingly used to study the binding of fluorescently-labeled peptides and proteins to phospholipid vesicles. In this work, we present a new method to analyze partition data obtained by this technique based on the assumption that the number of fluorescently-labeled protein molecules bound per liposome follows a Poisson distribution. To not overestimate the recovered partition coefficients, we first show that the variation in liposome brightness caused by this statistical distribution must be considered explicitly in data analysis when the parameter used to establish the partition curves is the fractional instead of the absolute amplitudes associated with the slowest diffusing particles in the system (lipid vesicles), a choice frequently made in FCS partition studies. We further extend the theoretical model describing the membrane partition of a fluorescently-labeled protein by considering the presence of a trace amount of free fluorescent dye (non-binding component) in the system. We show that this situation can account for an apparent maximal binding level lower than 100% in the experimental partitioning curves obtained for Alexa 488 fluorescently-labeled lysozyme and liposomes prepared with variable anionic phospholipid content. The extreme sensitivity of the FCS technique allowed uncoupling lysozyme partition from the protein-induced liposome aggregation, confirming that lysozyme binding to negatively charged liposomes is dominantly driven by electrostatic interactions.     

GABA interaction with lipids in organic medium

The interaction of 3H-GABA (gamma-aminobutyric acid and 14C-glutamate with lipids in an aqueous organic partition system was studied. With this partition system 3H-GABA and 14C-glutamate were able to interact with sphingomyelin, sulfatide, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and phosphatidic acid but not with cholesterol or ceramide. In an homogeneous aqueous medium we could not demonstrate any interaction between 3H-GABA and lipids. The apparent dissociation constants (Kd) for 3H-GABA-lipids or 14C-glutamate-lipids interactions in organic medium were in the millimolar range and maximal charge (Bmax) between 3 and 7 moles of GABA or glutamate by mole of lipid. Amino acids such as glutamic acid, beta-alanine and glycine displaced 3H-GABA with the same potency as GABA itself; thus these results show that the interaction lacks pharmacological specificity. To detect this interaction lipid concentrations higher than 2 microM were required and in the partition system 3H-GABA and lipid phosphorus were both concentrated at the interface. Therefore lipids tested with a biphasic partition system do not fulfill the classical criteria for a neurotransmitter receptor at least not for GABA and glutamate.     

Partition of proteins in aqueous two-phase systems containing charged dextran or hydrophobic PEG

Summary The electrochemical effect of a charged dextran derivative and the hydrophobic effect of hydrophobic chain PEG derivative on partitioning of six types of proteins in PEG/dextran aqueous two-phase systems were investigated- When 1. 6%(w/w)DEAE-dextran was present in the system,the partition coefficient decreased quickly with increasing pH value;when 0. 4% (w/w)PEG pentadecanoic acid ester was present in the system, the partition coefficient of protein with strong hydrophobicity was greatly increased. The experimental results show that the influence of hydrocarbon chain PEG derivative on partition coefficient is closely related to the hydrophobicity of proteins.     

Membrane surface properties of normal and malignant cells: partition in aqueous two-polymer phase systems

The partition of normal and malignantly transformed fibroblast lines and cell lines initiated from malignant human astrocytomas and a benign ganglioneuroma has been examined in aqueous dextran-polyethylene glycol phase system containing phosphate buffer with a low phosphate/sodium chloride ratio. The malignant astrocytomas showed a significantly lower partition coefficient as compared with the benign ganglioneuroma. Treatment of astrocytoma cells with dexamethasone caused an increase in the partitioning of the cell population. No differences were found in the partition behaviour of normal BHK-21 cells and their malignant transformants, the TRES fibrosarcoma cells. Polyoma and simian virus-transformed 3T3 fibroblasts showed partition ratios similar to the untransformed cells. Dexamethasone pre-treatment had no effect on the partition behaviour of these cells. The significance of these observations has been discussed in relation to the surface hydrophobicity and the neoplastic state.     

The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells

The P1 ParA protein is an ATPase that recognizes the parA promoter region where it acts to autoregulate the P1 parA–parB operon. The ParB protein is essential for plasmid partition and recognizes the cis -acting partition site parS . The regulatory role of ParA is also essential because a controlled level of ParB protein is critical for partition. However, we show that this regulatory activity is not the only role for ParA in partition. Efficient partition can be achieved without autoregulation as long as Par protein levels are kept within a range of low values. The properties of ParA mutants in these conditions showed that ParA is essential for some critical step in the partition process that is independent of par operon regulation. The putative nucleotide-binding site for the ParA ATPase was identified and disrupted by mutation. The resulting mutant was substantially defective for autoregulation and completely inactive for partition in a system in which the need for autoregulation is abolished. Thus, the ParA nucleotide-binding site appears to be necessary both for the repressor activity of ParA and for some essential step in the partition process itself. We propose that the nucleotide-bound form of the enzyme adopts a configuration that favours binding to the operator, but that the ATPase activity of ParA is required for some energetic step in partition of the plasmid copies to daughter cells.     

The partition system of multidrug resistance plasmid TP228 includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily

The segregational stability of bacterial, low-copy-number plasmids is promoted primarily by active partition. The plasmid-specified components of the prototypical P1 plasmid partition system consist of two proteins, ParA (44.3 kDa) and ParB (38.5 kDa), which, in conjunction with integration host factor, form a nucleoprotein complex at the plasmid partition site, parS. This complex is the probable substrate for the directed temporal and spatial intracellular movement of plasmids before cell division. The genetic organization of the partition cassette of the multidrug resistance plasmid TP228 differs markedly from that of the P1 paradigm. The TP228 system includes a novel member (ParF; 22.0 kDa) of the ParA superfamily of ATPases, of which the P1 ParA protein is the archetype. However, the ParF protein and its immediate relatives form a discrete subgroup of the ParA superfamily, which evolutionarily is more related to the MinD subgroup of cell division proteins than to ParA of P1. The TP228 and P1 partition modules differ further in that the former does not include a parB homologue, but does specify a protein (ParG; 8.6 kDa) unrelated to ParB. Homologues of the parF gene are widely disseminated on eubacterial genomes, suggesting that ParF-mediated partition may be a common mechanism by which plasmid segregational stability is achieved.     

CTP-controlled liquid–liquid phase separation of ParB

The ParABS system is supposed to be responsible for plasmid partitioning and chromosome segregation in bacteria. ParABS ensures a high degree of fidelity in inheritance by dividing the genetic material equally between daughter cells during cell division. However, the molecular mechanisms underlying the assembly of the partition complex, representing the core of the ParABS system, are still far from being understood. Here we demonstrate that the partition complex is formed via liquid–liquid phase separation. Assembly of the partition complex is initiated by the formation of oligomeric ParB species, which in turn are regulated by CTP-binding. Phase diagrams and in vivo analysis show how the partition complex can further be spatially regulated by parS. By investigating the phylogenetic variation in phase separation and its regulation by CTP, we find a high degree of evolutionary conservation among distantly related prokaryotes. These results advance the understanding of partition complex formation and regulation in general, by confirming and extending recently proposed models.     

P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.     

Detection of barbiturate-induced surface changes in cerebrocortical synaptosomes by phase partitioning in an aqueous two-phase system

The barbiturates tested in this work (barbital, phenobarbital, thiopental and pentobartial) modify the partition of synaptosomes in a Dextran T500-poly(ethylene glycol) 4000 two-phase system. Under adequate experimental conditions, the drugs increase the partition into the upper phase and this effect appears to be due to an action on the biological material and not on the interface potential of the system. This conclusion can be drawn from the fact that synaptosomes preincubated with low concentrations (0.1 mM) of barbital and pentobarbital maintained an increased partition into the upper phase. The extent of the effect observed appeared to be inversely proportional to the hydrophobicity of the drugs since phenobarbital and barbital showed a higher effect than thiopental and pentobarbital. Dithionite-induced anoxia, rotenone and ouabain also induced a similar increase of partition of synaptosomes into the upper phase, suggesting that the surface changes detected by phase partitioning modification of synaptosomes could be somehow related to the bioenergetic maintenance of the membrane ATPase.     

Heterogeneity in the surface properties of B16 melanoma cells from sublines with differing metastatic potential detected via two-polymer aqueous-phase partition

When mixed in aqueous solution at low concentrations, the neutral polymers dextran and poly(ethylene glycol) (PEG) rapidly form a two-phase system, consisting of a dextran-enriched lower phase and a PEG-enriched upper phase. Two B16 mouse melanoma cell lines, B16-F1 (low lung colonizing capability) and B16-F10 (high lung colonizing capability) were found to partition differentially into the upper phase in a variety of two-phase systems. Upper-phase partition depends primarily on either hydrophilic (i.e., surface charge density) or hydrophobic (i.e., affinity for the hydrocarbon chain of a PEG-fatty acid ester) cell surface properties, depending on the system used. In single-step partition studies, cells of the B16-F10 subline displayed a greater preference than B16-F1 cells for the upper phase in the hydrophilic system and less preference in systems sensitive to hydrophobic properties. Countercurrent distribution (CCD) experiments, performed with [125I]deoxyuridine DNA-labelled cells, were consistent with single-step partition results. These CCD results demonstrated that B16-F10 cells exhibited greater DNA synthesis than B16-F1 cells and that considerable heterogeneity, in both hydrophobic and hydrophilic surface properties, was present in subpopulations of cells of both sublines. The data also showed considerable enrichment of 125I-specific cell activity in certain sections of the distributions, indicating that differences in cellular DNA synthesis are reflected in the surface properties to which partition is sensitive.     

A plasmid partition system of the P1-P7par family from the pMT1 virulence plasmid of Yersinia pestis

The complete sequence of the virulence plasmid pMT1 of Yersinia pestis KIM5 revealed a region homologous to the plasmid partition (par) region of the P7 plasmid prophage of Escherichia coli. The essential genes parA and parB and the downstream partition site gene, parS, are highly conserved in sequence and organization. The pMT1parS site and the parA-parB operon were separately inserted into vectors that could be maintained in E. coli. A mini-P1 vector containing pMT1parS was stably maintained when the pMT1 ParA and ParB proteins were supplied in trans, showing that the pMT1par system is fully functional for plasmid partition in E. coli. The pMT1par system exerted a plasmid silencing activity similar to, but weaker than those of P7par and P1par. In spite of the high degree of similarity, especially to P7par, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1parS site, and the pMT1 Par proteins failed to support partition with P1parS or P7parS. Typical of other partition sites, supernumerary copies of pMT1parS exerted incompatibility toward plasmids supported by pMT1par. However, no interspecies incompatibility effect was observed between pMT1par, P7par, and P1par.     

Aqueous two-phase partition studies of the glucocorticoid receptor activation and pH-dependent changes of rat liver glucocorticoid-receptor complex in partly purified preparations

• 1.1. The activation of the glucocorticoid receptor of rat liver cytosol was studied by the use of partitioning in an aqueous dextran-poly(ethylene glycol) two-phase system.
  • 1.1.a, Incubation under conditions generally found to stimulate the activation results in a time-dependent increase of the partition coefficient of the glucocorticoid-receptor complex in the two-phase system.
  • 1.2.b, This increase was found to result from the fact that the activated form of the complex has a higher partition coefficient that the nonactivated one, since the two forms are eluted from DNA-Sepharose columns as clearly separated peaks with different partition coefficients, and a redistribution of complex between the peak occurs without any changes of the partition coefficient of each single peak.
  • 1.3.c, The partition coefficient of a partly purified nonactivated complex was found to follow the same time course towards a higher value under ‘activating’ incubation conditions as the complex in whole cytosol.
• 2.2. The dependence of the partition coefficient of the glucocorticoid-receptor complex on the pH of the two-phase system revealed a pH-dependence of the state of the complex. Experiments with partly purified preparations of the complex showed that this change was either a conformational change or an aggregation of high affinity of the complex with itself or another DNA-binding substance.

     

Characterization of Ternary Protein Systems In?Vivo with Tricolor Heterospecies Partition Analysis

Tools and assays that characterize protein-protein interactions are of fundamental importance to biology, because protein assemblies play a critical role in the control and regulation of nearly every cellular process. The availability of fluorescent proteins has facilitated the direct and real-time observation of protein-protein interactions inside living cells, but existing methods are mostly limited to binary interactions between two proteins. Because of the scarcity of techniques capable of identifying ternary interactions, we developed tricolor heterospecies partition analysis. The technique is based on brightness analysis of fluorescence fluctuations from three fluorescent proteins that serve as protein labels. We identified three fluorescent proteins suitable for tricolor brightness experiments. In addition, we developed the theory of identifying interactions in a ternary protein system using tricolor heterospecies partition analysis. The theory was verified by experiments on well-characterized protein systems. A graphical representation of the heterospecies partition data was introduced to visualize interactions in ternary protein systems. Lastly, we performed fluorescence fluctuation experiments on cells expressing a coactivator and two nuclear receptors and applied heterospecies partition analysis to explore the interactions of this ternary protein system.     

Kinetic theory of enzymatic reactions in reversed micellar systems. Application of the pseudophase approach for partitioning substrates

A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving substrates capable of partitioning among all pseudophases of the micellar system i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system.