Aqueous Two-phase Separation Research Articles

(9,8) Single‐Walled Carbon Nanotube Enrichment via Aqueous Two‐Phase Separation and Their Thin‐Film Transistor Applications

One-step simultaneous diameter and metallicity enrichment of semiconducting (9,8) single-walled carbon nanotubes is achieved by a new aqueous two-phase separation method. The enriched nanotubes have a band gap of 0.88 eV and a narrow emission window at 1400 nm, showing excellent potential for application in electronic and optoelectronic devices. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Bench-top aqueous two-phase extraction of isolated individual single-walled carbon nanotubes

Isolation and purification of single-walled carbon nanotubes (SWCNTs) are prerequisites for their implementation in various applications. In this work, we present a fast (∼5 min), low-cost, and easily scalable bench-top approach to the extraction of high-quality isolated SWCNTs from bundles and impurities in an aqueous dispersion. The extraction procedure, based on aqueous two-phase (ATP) separation, is widely applicable to any SWCNT source (tested on samples up to 1.7 nm in diameter) and independent of defect density, purity, diameter, and length. The extracted dispersions demonstrate that the removal of large aggregates, small bundles, and impurities is comparable to that by density gradient ultracentrifugation, but without the need for high-end instrumentation. Raman and fluorescence-excitation spectroscopy, single-nanotube fluorescence imaging, atomic force and transmission electron microscopy, and thermogravimetric analysis all confirm the high purity of the isolated SWCNTs. By predispersing the SWCNTs without sonication (only gentle stirring), full-length, pristine SWCNTs can be isolated (tested up to 20 μm). Hence, this simple ATP method will find immediate application in the generation of SWCNT materials for all levels of nanotube research and applications, from fundamental studies to high-performance devices.

Latent and active aurone synthase from petals of C. grandiflora: a polyphenol oxidase with unique characteristics.

Main conclusion Aurone synthase belongs to the novel group 2 polyphenol oxidases and the presented kinetic characterization suggests a differing aurone biosynthesis in Asteraceae species compared to snapdragon. Aurone synthases (AUS) are polyphenol oxidases (PPO) physiologically involved in the formation of yellow aurone pigments in petals of various Asteraceae species. They catalyze the oxidative conversion of chalcones into aurones. Latent (58.9 kDa) and active (41.6 kDa) aurone synthase from petals of C. grandiflora was purified by a quantitative removal of pigments using aqueous two-phase separation and several subsequent chromatographic steps. The purified enzymes were identified as cgAUS1 (A0A075DN54) and sequence analysis revealed that cgAUS1 is a member of a new group of plant PPOs. Mass determination experiments of intact cgAUS1 gave evidence that the C-terminal domain, usually shielding the active site of latent polyphenol oxidases, is linked to the main core by a disulfide bond. This is a novel and unique structural feature of plant PPOs. Proteolytic activation in vivo leads to active aurone synthase possessing a residual peptide of the C-terminal domain. Kinetic characterization of purified cgAUS1 strongly suggests a specific involvement in 4-deoxyaurone biosynthesis in Coreopsis grandiflora (Asteraceae) that differs in various aspects compared to the 4-hydroxyaurone formation in Antirrhinum majus (Plantaginaceae): cgAUS1 is predicted to be localized in the thylakoid lumen, it possesses exclusively diphenolase activity and the results suggest that aurone formation occurs at the level of chalcone aglycones. The latent enzyme exhibits allosteric activation which changes at a specific product concentration to a constant reaction rate. The presented novel structural and functional properties of aurone synthase provide further insights in the diversity and role of plant PPOs.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2261-0) contains supplementary material, which is available to authorized users.

High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae

Wheat germ agglutinin (WGA) is a homodimeric lectin stabilized by non-covalent interactions. Each monomer of 171 residues, which has a complex structure with 16 disulfide bridges, determines two sites for the specific binding of N-acetyl-d-glucosamine and one for the specific binding of N-acetylneuraminic acid. Because of these folding requirements, the production of high yields of recombinant WGA is still not possible and its extraction from wheat germ is the only source for commercial purposes. This work reports for the first time the expression of WGA isolectin A (WGA A), using a baculovirus expression system in Sf9 cells and Rachiplusia nu larvae. High levels of recombinant WGA A were obtained in both cases, especially in R. nu, where yields reached 346.6±88.5μg/g of larvae. Also, an integrated purification method was developed based on aqueous two-phase separation coupled to affinity chromatography using chitosan mini-spheres. The recombinant WGA A was able to recognize ovoalbumin sugar moieties, cross-react with anti-WGA serum and agglutinate human red blood cells, and showed the same behavior as that of commercial WGA in SDS-PAGE and RP-HPLC analyses.

Surfactants' Key Role in Ionic Aqueous Two-Phase Separation of Carbon Nanotubes Towards Simple Chirality Isolation

SWCNTs are known to have wide distribution in diameter/chirality when produced; leading to variability in the starting material chirality distributions with respect to production batch and method. Therefore numerous separation methods that provide nearly single-chiralities have been reported. Most of these separation techniques involved numerous iterations to achieve high purity (gel chromatography, DGU) and long separation times, thereby preventing low-cost scalability of the method. Recently aqueous two-phase (ATP) separation has been explored as a new promising method to separate single-wall carbon nanotubes (SWCNTs) by diameter. In this work, we determined that the ATP separation mechanism is driven by the hydrophobicity of the surfactant, or combination of surfactants, covering the SWCNTs, which can be tuned by mixing different surfactants in well-defined ratios. Having a three-surfactant system, with very different diameter-dependent affinities for SWCNTs, and the combination of salt to adjust the specific surfactant structure surrounding the SWCNTs, has allowed us to develop a simple strategy for obtaining highly enriched single-chirality suspensions of (6,5) SWCNTs in only 1 or 2 steps, thereby obtaining a high separation yield and simplifying the separation technique. Single chirality solutions can now be obtained in an extremely fast, and easy-to-scale-up, cheap way.

Role of Surfactants and Salt in Aqueous Two-Phase Separation of Carbon Nanotubes toward Simple Chirality Isolation

Aqueous two-phase extraction has recently been demonstrated as a new method to separate single-wall carbon nanotubes (SWCNTs). In this work, we determined that the mechanism of separation is driven by the hydrophobicity of the surfactant, or combination of surfactants, at the SWCNT surface. This knowledge allowed us to develop a simple approach for obtaining highly enriched single-chirality suspensions in only 1 or 2 steps. These results were obtained by strategically combining multiple surfactants with different diameter-dependent binding affinities for SWCNTs and salts that readjust the surfactant structure within the mixed micelle surrounding the SWCNTs. The procedure is successfully applied to SWCNTs from different sources (CoMoCAT and HiPco) with various diameter distributions (from 0.53 to 1.2 nm). Each separation step is characterized by optical absorption, resonant Raman, and photoluminescence excitation spectroscopies. By determining the SWCNT sorting mechanism, we were able to develop a new set of parameters that separated another chirality.

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low-cost purification by surfactant-based aqueous two-phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY-2) suspension cell platform and the establishment of pilot-scale propagation and downstream processing including first-step purification by ATPS. Green fluorescent protein-hydrophobin fusion (GFP-HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY-2 suspension cells was scaled up in standard stirred tank bioreactors up to 600L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP-HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20L volume, without loss off efficiency. This study provides the first proof of concept for large-scale hydrophobin-assisted production of recombinant proteins in tobacco BY-2 cell suspensions.

The influence of inorganic salts on the rheological properties of 1,3-propanediyl bis(dodecyl dimethylammonium bromide) and sodium dodecylsulfonate aqueous mixed system

The rheological properties of isotropic 12-3-12/AS/H2O mixed systems in the absence and in the presence of salt have been investigated. The addition of AS into 12-3-12 aqueous solution or vice versa, or the addition of salt into isotropic 12-3-12/AS/H2O mixed systems, leads to changes in microstructure, phase behavior, shear viscosity η, and rheological behavior. In the process of gradually adding AS (or salt) to 12-3-12 aqueous solution, the microstructural transitions are as follows: coexistence of spherical micelles and rodlike micelles→somewhat elongation of rodlike micelles→coexistence of rodlike micelles and vesicles→vesicles; and the rheological behaviors change as follows: antithixotropic and shear thickening→complex thixotropic→shear thinning and thixotropic. η increases initially to a peak, then decreases, and finally the further addition of AS or salt results in aqueous two-phase separation. The origin and the small value (∼510mPas) of the η peak for isotropic 12-3-12/AS/H2O mixed system with excess 12-3-12 are linked with the above microstructural transitions. The η peak becomes lower with the addition of salt. The salt effect on the η peak is related to the matching of water affinities between the counterions and ionic head groups of surfactant. The isotropic 12-3-12/AS/H2O mixed systems with excess AS are Newtonian fluids or near Newtonian fluids with η≈0.75mPas under the shear rate of 1500s−1 due to the coexistence of spherical micelles and short rodlike micelles. The addition of salt changes these Newtonian or near Newtonian fluids to antithixotropic fluids, or complex thixotropic fluids, or thixotropic fluids, and leads to somewhat increase of η (usually less than 25mPas). Two-parameter power-law is used to model the above mentioned fluids. Most of the fluids excepting a few shear thinning fluids located near the phase boundary between isotropic single-phase region and adjacent aqueous two-phase region obey the power-law model.

Phase Behavior and Molecular Dynamics Simulation Studies of New Aqueous Two-Phase Separation Systems Induced by HEPES Buffer

Here, for the first time, we show that with addition of a biological buffer, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), into aqueous solutions of tetrahydrofuran (THF), 1,3-dioxolane, 1,4-dioxane, 1-propanol, 2-propanol, tert-butanol, acetonitrile, or acetone, the organic solvent can be excluded from water to form a new liquid phase. The phase diagrams have been determined at ambient temperature. In order to understand why and how a zwitterion solute (HEPES) induced phase separation of the investigated systems, molecular dynamics (MD) simulation studies are performed for HEPES + water + THF system. The MD simulations were conducted for the aqueous mixtures with 12 different compositions. The reliability of the simulation results of HEPES in pure water and beyond the phase separation mixtures was justified by comparing the densities obtained from MD with the experimental values. The simulation results of HEPES in pure THF and in a composition inside the phase separation region were justified qualitatively. Interestingly, all HEPES molecules entirely aggregated in pure THF. This reveals that HEPES is insoluble in pure THF, which is consistent with the experimental results. Even more interestingly, the MD simulation for the mixture with composition inside the phase separation region showed the formation of two phases. The THF molecules are squeezed out from the water network into a new liquid phase. The hydrogen bonds (HBs), HB lifetime, HB Gibbs energy (ΔG), radial distribution functions (RDFs), coordination numbers (CNs), electrostatic interactions, and the van der Waals interactions between the different species have been analyzed. Further, MD simulations for the other phase separation systems by choosing a composition inside the two liquids region for each system were also simulated. Our findings will therefore pave the way for designing new benign separation auxiliary agents.

Electrostatic Interactions and Aqueous Two-Phase Separation Modes of Aqueous Mixed Oppositely Charged Surfactants System

Electrostatic interactions play an important role in setting the aqueous two-phase separation behaviors of mixtures of oppositely charged surfactants. The aqueous mixture of cetyltrimethylammonium bromide (CTAB) and sodium dodecylsulfonate (AS) is actually a five-component system, comprised of CTAB, AS, complex salt (cetyltrimethylammonium dodecylsulfonate, abbreviated as CTA(+)AS(-)), NaBr, and water. In the three-dimensional pyramid phase diagram, the aqueous two-phase region with excess AS or with excess CTAB extends successively from the region very near to the NaBr-H2O line through the CTAB-AS-H2O conventional mixing plane to the CTA(+)AS(-)-AS-H2O side plane or to the CTA(+)AS(-)-CTAB-H2O side plane, respectively. Large or small molar ratios between the counterions and their corresponding surfactant ions for oppositely charged surfactants located in the NaBr side or the CTA(+)AS(-) side of the pyramid imply strong or weak electrostatic screening. Electrostatic screening of counterions alters the electrostatic attractions between the oppositely charged head groups or the electrostatic repulsions between the like-charged head groups in excess, and the electrostatic free energy of aggregation thus affects the aqueous two-phase separation modes. Composition analysis, rheological property investigation, and TEM images suggest that there are two kinds of aqueous two-phase systems (ATPSs). On the basis of these experimental results and Kaler's cell model, two kinds of phase separation modes were proposed. Experimental results also indicate that all of the top phases are surfactant-rich, and all of the bottom phases are surfactant-poor; the density difference between the top phase and the bottom phase in one ATPS is very small; the interfacial tension (σ) of the ATPS is ultralow.

Trends in whey protein fractionation

Whey is a by-product of cheese manufacture that is normally treated as a waste. However, it contains a mixture of proteins with important nutritional and biological attributes. To extract these valuable proteins, whey fractionation has been developed using three main techniques; namely chromatographic (e.g., ion-exchange and hydrophobic adsorption), membrane (e.g., traditional pressure-driven and electro-separation)-, or combined methods. Recently, new promising techniques have been introduced such as aqueous two-phase separation (ATPS) and magnetic fishing. This article reviews the use of these techniques together with an evaluation of their performance regarding the yield and purity of two major proteins in whey.

Salt effect on the rheological properties of the aqueous mixed cationic and anionic surfactant systems

The effect of NaBr on the aqueous two-phase regions of 0.10 mol kg −1 cetyltrimethylammonium bromide (CTAB)/sodium dodecyl sulfonate (AS)/H 2O mixed system at 318.15 K has been investigated. The aqueous two-phase region with excess AS or the aqueous two-phase region with excess CTAB all moves far from the equimolar line with the addition of NaBr. The effect of NaBr on the shear viscosities of the systems at the two isotropic single-phase regions has been also studied. At each isotropic single-phase region, one shear viscosity peak was observed for each m NaBr. The addition of NaBr to the mixed CTAB/AS/H 2O systems shifts the viscosity peaks away from the equimolar line, has stronger effect on the viscosity peak at the isotropic single-phase region with excess CTAB, and weaker effect on that with excess AS. Seven 0.10 mol kg −1 CTAB/AS/H 2O isotropic systems adjacent to the viscosity peaks were chosen to investigate the effect of NaBr on the shear viscosity. The rheological behaviors as well as the shear viscosity values of the seven chosen systems all change with the addition of NaBr. Salt-induced aqueous two-phase systems can be obtained with the addition of NaBr to the seven chosen systems. Just before the salt-induced aqueous two-phase separation was observed, the common tendency of the seven chosen systems is the decreases of the shear viscosities with the addition of NaBr, and those isotropic systems with NaBr aqueous solutions as solvents and very close to the phase boundaries are all thixotropic fluids. These results indicate that the salt-induced aqueous two-phase systems originate from thixotropic isotropic systems.

Analysis of the Formation Mechanism for Thermoresponsive-Type Coacervate with Functional Copolymers Consisting of N-Isopropylacrylamide and 2-Hydroxyisopropylacrylamide

We now report the formation mechanism of the thermoresponsive-type coacervate with the novel functional temperature-sensitive polymer, poly(N-isopropylacrylamide-co-2-hydroxyisopropylacrylamide) (poly(NIPAAm-co-HIPAAm)), synthesized in our laboratory. The effects of introducing the hydrophilic comonomer (HIPAAm) into the copolymer chains and adding salts on the behaviors of the coacervate droplets induced in the poly(NIPAAm-co-HIPAAm) aqueous solutions were investigated. Not only the particle sizes of the coacervate droplets but also the cloud points of the copolymer solutions could be modulated by the HIPAAm content incorporated in the copolymers. Moreover, the particle sizes of the coacervate droplets were also changed by adding salts. Namely, the particle sizes increased with the decreasing HIPAAm composition and increasing NaCl concentration. In addition, the 1H NMR and differential scanning calorimetric measurements suggested that as the HIPAAm content decreased or NaCl concentration increased, dehydration of the copolymers induced in the phase transition and/or separation became much easier. Therefore, on the basis of the findings obtained from these measurements, we determined that the particle sizes of the coacervate droplets induced in the temperature-sensitive polymers increased as the number of the water molecules, which are dissociated from the polymeric chains during the phase transition and/or separation, increased. Besides, to examine the separation of the model solutes, the aqueous two-phase separation with the coacervate droplets of poly(NIPAAm-co-HIPAAm) was carried out. The partitions of Methyl Orange as a model solute under both acidic (pH 2) and basic (pH 12) conditions were performed. The amount of Methyl Orange partitioned into the coacervate droplets at pH 12 is much greater than that at pH 2, which indicated that the coacervate droplets could recognize a slight difference in the polarity or structure between the model solutes.

Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn(2+) cations and a K(m) of 0.15 and a V(max) of 0.47 nmol.min(-1).mg(-1) protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.

A generalized approach to thermodynamic properties of biomolecules for use in bioseparation process design

Bioseparation techniques exploit the differences of physicochemical or thermodynamic properties between the product and the contaminants. Rapid development of a downstream process, therefore, requires physicochemical and thermodynamic characterization of the components to be separated. In this paper, we investigate whether a generalized thermodynamic interrelation exists among different parameters. For instance activity coefficients, osmotic virial coefficients and the solubility of macromolecules are interrelated to each other. Experimental determination of any one of these parameters can be translated across the boundaries of different separation techniques. A number of downstream separation processes, including size-exclusion chromatography, hydrophobic-interaction chromatography, reversed-phase chromatography, aqueous two-phase separation, crystallization and precipitation, are found to be explained and designed using this generalized thermodynamics. This generalization of thermodynamic properties together with high-throughput experimentation provides a systematic and high-speed approach to bioseparation process development and optimization. The applicability of this approach for bioseparation process design was investigated by a case study on nystatin, a medium-sized biomolecule. The distribution coefficients of nystatin in reversed-phase chromatography showed straightforward relationship with the solubilities at various solvent compositions and the experimental data supported the trend of the relationship.

Protein production and induction of the unfolded protein response inTrichoderma reeseistrain Rut-C30 and its transformant expressing endoglucanase I with a hydrophobic tag

The effect of induction of protein production was studied in bioreactor cultures of T. reesei strain Rut-C30 and its transformant expressing endoglucanase I core domain (EGI, Cel7B) fused with a hydrophobic peptide tag. The tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The fungi were first grown on glucose-containing minimal medium after which rich medium with lactose as a carbon source was added to induce cellulase production. Production of extracellular protein and cellulase activity and the transcript levels of the major cellulase genes were analyzed during the cultivations. Induction of the cellulase genes followed a similar temporal pattern in both strains. The first phase of induction took place after addition of lactose as soon as glucose was depleted, and the second phase after lactose was consumed. Western analysis showed that a decreased amount of fusion protein was produced in the culture medium compared with the endogenous EGI, although the strain harbors several copies of the recombinant gene under the strong cbh1 promoter. The fusion protein appeared to accumulate within the cells, indicating impaired secretion of the protein. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the active form of hac1 transcript encoding the UPR transcription factor increased concurrently with induction of the cellulase genes in both strains, indicating increased requirement of the folding machinery under these conditions. However, only a minor increase in bip1 and pdi1 transcript level was observed in the transformant compared with the parental strain.

Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems.

In this work we describe the new concept of using fungal hydrophobins as efficient tags for purification of recombinant fusion proteins by aqueous two-phase separation. Hydrophobins are a group of small surface-active proteins produced by filamentous fungi. Some characteristics of hydrophobins are that they are relatively small (approximately 100 amino acids), they contain eight disulfide-forming Cys residues in a conserved pattern, and they self-assemble on interfaces. The aqueous two-phase systems studied were based on nonionic surfactants that phase-separate at certain temperatures. We show that the use of hydrophobins as tags has many advantages such as high selectivity and good yield and is technically very simple to perform. Fusion proteins with target proteins of different molecular size were compared to the corresponding free proteins using a set of different surfactants. This gave an understanding on which factors influence the separation and what rationale should be used for optimization. This unusually strong and specific interaction between polymeric surfactants and a soluble protein shows promise for new developments in interfacing proteins and nonbiological materials for other applications as well.

Reversibly soluble macroaffinity ligand in aqueous two-phase separation of enzymes

Use of alginate as a free bioligand incorporated in an aqueous two-phase system of polyethylene glycol 6000–salt resulted in considerable purification of wheat germ α-amylase and sweet potato β-amylase from their crude extracts. The elution of the enzyme from the free bioligand was facilitated by exploiting the fact that alginate can be reversibly precipitated in the presence of Ca 2+. α-Amylase could be purified 42-fold with 92% activity recovery. β-Amylase on the other hand could be purified 43-fold with 90% recovery. Both purified enzymes showed a single band on sodium dodecylsulfate–polyacrylamide gel electrophoresis.

Rapid process for purification of an extracellular β-xylosidase by aqueous two-phase extraction

A rapid process for purification of an extracellular β-xylosidase with high purity was developed. The manipulation involved the precipitation of protein from culture medium and the extraction of enzyme from the resuspended crude protein solution by an aqueous-two phase separation. A linear random copolymer, PE62, with 20% ethylene oxide and 80% propylene oxide was employed in both stages of the purification. The enzyme was precipitated effectively by using 10% (w/v) PE62 and 5% (w/v) Na 2HPO 4. The aqueous two-phase extraction was performed with PE62 (10%)–NaH 2PO 4 (15%) as phase-forming reagent. SDS–PAGE analysis revealed that the purified enzyme is near homogeneity. The yield is about 100% with a purification factor of 8.8-fold. The whole process can be completed within an hour without any column chromatography.

Binding Affinity of the Methyl Ester of AK-toxin I to Membrane Fractions from Japanese Pear Leaves

The binding site of AK-toxin, a host-specific toxin against Japanese pear, was searched for in the membrane fractions of the pear leaves, using 3H-labeled AK-toxin I methyl ester. Binding activity, which was displaceable by the unlabeled ligand, was observed for microsomal fraction from a toxin-susceptible cultivar, Nijisseiki. However, the binding was also observed for those from toxin-resistant cultivars, Kosui and Hosui. Detection of the specific binding failed for the plasma membrane fraction which was prepared from microsomal fraction of the toxin-susceptible cultivar by aqueous two-phase separation, and the hitherto presumed model of the AK-toxin receptor in the plasma membrane could not be verified.