Purification of rat C6 glioma plasma membranes by affinity partitioning

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.     

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

Partitioning in dextran–poly(ethylene)glycol (PEG) aqueous–aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum<mitochondria<Golgi apparatus<lysosomes and endosomes<plasma membranes. Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.     

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin-dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin-dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.     

An aqueous hydroxypropylstarch-poly(ethylene glycol) two-phase system for extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey.

The partitioning of alpha-lactalbumin and beta-lactoglobulin from bovine whey has been studied in an aqueous poly(ethylene glycol) (PEG)-hydroxypropylstarch two-phase system. The influence of several parameters including concentrations of polymers, sodium phosphate buffer, KSCN, and of PEG palmitate, with and without the presence of Ca2+, on the partitioning of the proteins has been investigated. The separation of the two proteins was demonstrated by counter-current distribution. A purification procedure for both proteins has been developed by using PEG-hydroxypropylstarch two-phase system. This system is compared with the more costly standard system based on PEG and dextran. The possible use of the aqueous two-phase systems for batch extraction for large scale purification of these whey proteins is discussed.     

Heterogeneity of synaptosomal membrane preparations from different regions of calf brain studied by partitioning and counter-current distribution.

1. Membranes obtained by lysis and Yeda-press treatment of synaptosomes (nerve endings) from cortex, caudateus nucleus, and hippocampal region of calf brain have been studied by partitioning within a liquid-liquid aqueous two-phase system consisting of water, dextran, Ficoll, and poly(ethylene glycol). 2. The partitioning of membranes was sensitive to the presence of a dextran-bound dye, Procion yellow HE-3G, in the lower phase. 3. The two-phase system was used for counter-current distribution to study the heterogeneity of the synaptic membranes from the three regions of the brain and to separate the membranes into fractions. 4. The obtained counter-current distribution profiles strongly depended on the region of the brain from which the membranes were isolated. 5. The membrane fractions obtained showed marked differences in their SDS electrophoresis pattern.     

Selective protein separations using Formed-In-Place anion exchange membranes.

Anion exchange membranes prepared by adsorption of polymers on Formed-In-Place microfiltration substrates were formed and ion-exchange separations of solutions containing two proteins were determined by ion exchange membrane sequential separation procedures, similar to affinity membrane separation procedures. Representative ion exchange separation processes utilizing adsorbed poly(ethylene imine) (PEI) as the ion exchange membrane for the separation of the components of solutions containing two proteins, bovine serum albumin (BSA) and lysozyme and ovalbumin and lysozyme, are described. The stability of the PEI adsorbed layer, binding characteristics of the BSA to the membrane and purification properties of the procedure were determined.     

Extraction of proteins from material rich in anionic mucilages: partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems

For various reasons extraction of proteins from plant material is difficult. In particular phenolic compounds and polyanionic cell-wall mucilages render conventional procedures of extraction and purification much more difficult. In this respect, aqueous polymer two-phase systems are presented as a powerful technique in extraction of vanadate-dependent bromoperoxidases from the brown macroalga Laminaria digitata, a seaweed extremely rich in mucilages. Little bromoperoxidase activity was obtained when fresh thallus material was extracted in Tris buffer. Extraction from freeze-dried and powdered material was more efficient but only satisfactory when partitioning in an aqueous polymer two-phase system was employed. Among several two-phase systems tested, one composed of poly(ethylene glycol) (PEG 1550) and potassium carbonate proved most successful (phase system-1). A rapid and efficient extraction procedure was developed with special regard for suitability in large scale processes. Staining for catalytic activity after PAGE revealed a pattern of several bromoperoxidase isoforms. Bromoperoxidases extracted in phase system-1 were fractionated into two groups of isoforms by partitioning in a second system (phase system-2) indicating that isoforms from both groups differ significantly in surface properties. Subsequently, one purification step by hydrophobic interaction chromatography was sufficient to remove residual non-peroxidase proteins as well as remaining polysaccharides from bromoperoxidases of both groups. Thus, consideration of aqueous two-phase systems as a technique for extraction and purification of plant proteins can be recommended, whenever inconveniant amounts of phenolic compounds, mucilages or pigments are present.     

Orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor. Fluorescence studies

Four flourescein isothiocyanate (FITC) derivatives of Naja naja siamemsis 3 neurotoxin (alpha-toxin), labeled at the epsilon-amino groups of Lys-23, Lys-35, Lys-49, or Lys-69, and a tetramethylrhodamine isothiocyanate (TRITC) derivative, labeled at epsilon-amino group of Lys-23, were prepared and used to analyze the orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor (AcChR) relative to both the plane of the membrane and the central ion channel. Fluorescence-quenching studies of the AcChR-bound FITC derivatives indicated significant solute accessibility to each site of labeling and suggested that none of the sites of FITC labeling is included in the binding surface of the alpha-toxin. Labeling of Lys-23 with TRITC did not affect the affinity of the alpha-toxin toward the AcChR and confirmed, contrary to some previous reports, a minimal role of Lys-23 in the binding surface of the alpha-toxin. Measurements of energy transfer between the lipid-membrane surface and the sites of labeling on receptor-bound alpha-toxin derivatives show that the relative distances of closest approach between the surface of the lipid membrane domain and the sites of labeling are in the order Lys-23 less than or equal to Lys-49 less than Lys-35 less than or equal to Lys-69. Energy transfer between AcChR tryptophans and the sites of labeling of bound derivatives was about 50% greater to Lys-49 than to Lys-23, Lys-35, or Lys-69, suggesting that Lys-49 is closer to receptor tryptophans and to the center of the extracellular domain of the receptor than Lys-23, Lys-35, or Lys-69. Combined with previous observations that the tip of the central loop of the alpha-toxin directly interacts with the AcChR, the above results suggest a model of the approximate orientation of the snake neurotoxins on the receptor. This model shows the tip of the central loop of the toxin directly interacting with the receptor surface and the major axis of the neurotoxin tilting from a perpendicular projection from the membrane. The surface of the alpha-toxin that includes Lys-23 projects away from the central ion channel and the surface that includes Lys-35 and Lys-69 faces the ion channel.     

Recovery of extracellular human insulin-like growth factor-I and II as a fusion protein from Escherichia coli culture broth by aqueous two-phase extraction.

The primary purification of human insulin-like growth factor-I (IGF-I) and IGF-II, produced extracellularly in Escherichia coli as a fusion to two domains (ZZ) derived from staphylococcal protein A, has been studied. First, the partitioning of IgG-affinity purified ZZ-IGF-I and ZZ-IGF-II, respectively, to the top phase in poly(ethylene glycol)/potassium phosphate aqueous two-phase systems were investigated. Thereafter, the extraction of ZZ-IGF-I with a poly(ethylene glycol) 1500/potassium phosphate system was performed directly in the bioreactor after the cultivation. This resulted in a reduction of the cultivation volume more than 3-fold with a recovery of about 90% of target protein in a poly(ethylene glycol)-rich phase. The majority of the cells partitioned to the potassium phosphate-rich bottom phase, while a smaller fraction was collected at the interface, and/or as a densely packed cake on top of the interface. Contaminating proteins were also eliminated to some extent, which resulted in an almost 2-fold protein purification. Some obvious benefits offered by the aqueous two-phase system in the primary purification have been demonstrated: Firstly, the possibility to an early process volume reduction and thereby a concentration of the target protein. Secondly, a simultaneous protein purification was achieved. From this work it can be concluded that aqueous two-phase extraction should be considered as an attractive candidate for the primary steps during the design of new purification processes for extracellular proteins.     

Permeability studies in chitosan membranes. Effects of crosslinking and poly(ethylene oxide) addition

Pure chitosan, glutaraldehyde crosslinked chitosan, and a blend of chitosan with poly(ethylene oxide) (PEO) membranes were prepared. The three membranes were characterized in terms of their swelling capacities as well as their permeabilities to a drug model (sulfamerazine sodium salt). For the permeation experiments, the variables analyzed were the type of membrane and the initial drug concentration in the liquid phase (from 0.1% to 1.5%). Permeability coefficients were calculated using UV spectroscopy. The results showed that for the three analyzed membranes, the permeability did not change with time (over the studied time interval). An increase in the permeability for CHI/PEO membranes compared to those made of pure chitosan was also observed, possibly due to microporous region formation and/or crystallinity reduction. For the crosslinked membrane, an even higher increase in the permeability coefficient was observed. In this case, the increase was attributed to free volume variation.     

Aqueous polymer two-phase systems formed by new thermoseparating polymers

A set of new polymers that can be used as phase forming components in aqueous two-phase systems is presented. All polymers studied have thermoseparating properties i.e. form one separate polymer enriched phase and one aqueous solution when heated above the critical temperature. This property makes the polymers attractive alternatives to the polymers used in traditional aqueous two-phase systems such as poly(ethylene glycol) (PEG) and dextran. The thermal phase separation simplifies recycling of the polymers, thus making the aqueous two-phase systems more cost efficient and suitable for use in large scale. Thermoseparating polymers studied have been copolymers of ethylene oxide and propylene oxide (EO-PO), poly (N-isopropylacrylamide) (poly-NIPAM), poly vinyl caprolactam (poly-VCL) and copolymers of N-isopropylacrylamide and vinyl caprolactam with vinyl imidazole (poly(NIPAM-VI) and poly(VCL-VI), respectively). In addition, the copolymer poly(NIPAM-VI) has the property to be uncharged at pH above 7.0 and positively charged at lower pH. This allows the partitioning of protein to be directed by changing the pH in the system instead of the traditional addition of salt to direct the partitioning. Hydrophobically modified EO-PO copolymer (HM-(EO-PO)) with alkyl groups (C₁₄) at both ends forms two-phase system with for example poly(NIPAM-VI). The phase diagram for poly(NIPAM-VI)/HM-(EO-PO) was determined and the model proteins lysozyme and BSA were partitioned in this system. For BSA in poly(NIPAM-VI)/HM-(EO-PO) system a change in pH from 8.0 to 5.4 results in a change of partition coefficient from K=0.8 to K=5.1, i.e. BSA could be transferred from the HM-(EO-PO) phase to the poly(NIPAM-VI) phase. BSA partitioning in poly(NIPAM-VI)/HM-(EO-PO) system allows quantitative BSA recovery, and recoveries of poly(NIPAM-VI) and HM-(EO-PO) were 53% and 92%, respectively, after the thermoseparation step.     

Yeast cell debris and protein partitioning in the poly(ethylene glycol)-poly(vinyl alcohol) biphasic system

Yeast cells, cell debris and protein partitioning have been investigated in the poly(ethylene glycol) (PEG) 8000/poly(vinyl alcohol) (PVA) 10,000 system. Cells and cell debris partition into the lower (PVA) phase over the pH range 4.8-7.5, and with up to 0.37 M KCl at pH 5.9. Protein partitioning is more pH-dependent in the PEG/PVA system than in the PEG/dextran system, and a significant fraction of the total protein is found at the interface at lower pH values. Significant, rapid purification of overproduced pyruvate kinase in a PEG/PVA system containing Blue Sepharose CL-6B particles is demonstrated.     

Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.     

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.     

Synaptic membranes from Torpedo marmorata electric organ. 1. Separation and analysis of nicotinic acetylcholine receptor- and acetylcholinesterase-containing membrane vesicles using aqueous two-phase systems

Two-phase systems consisting of water, dextran and poly(ethylene glycol) have been used for partition of membranes obtained from Torpedo marmorata electric organ. The partition behaviour of the membranes could be adjusted by using a polymer with covalently-bound charged groups. By using this method, the membranes were divided into several fractions which were analyzed for nicotinic acetylcholine receptor and acetylcholinesterase content. It was found that nicotinic receptor-enriched membranes were separated from those containing esterase in a single partition step. Receptor-enriched membranes obtained by gradient centrifugation could be further separated into two receptor fractions by the two-phase technique. The results also reveal at least two types of acetylcholinesterase-rich membranes.     

Preparation and characterization of muscarinic-acetylcholine-receptor-enriched membranes from pig atria.

A procedure was developed for the large scale preparation of membranes from pig atria which are enriched 10-13 fold in the muscarinic acetylcholine receptor. The procedure involved differential centrifugation and sucrose-gradient centrifugation in solutions containing 150 mM-NaClO4 and 5 mM-EDTA to minimize membrane aggregation. The final membrane preparation bound about 1.1 pmol of L-quinuclidinyl benzilate/mg of protein. Comparable results were obtained with either fresh or frozen tissue. About the same yield (120 pmol of L-quinuclidinyl benzilate sites/100 g of tissue) and specific activity of membranes were obtained from different regions of the atria. The final preparation was stable at -80 degrees C in buffered sucrose solutions. The membranes appeared mostly as sheets or fragments and partly as closed vesicles in the electron microscope and were heterogeneous in isopycnic Percoll gradients. Marker enzyme studies showed that the receptor was enriched in parallel with the plasma membrane markers guanylate cyclase (particulate form) and (Na+ + K+)-activated ATPase. Some contamination by mitochondrial outer and endoplasmic reticulum membranes was evident from the distribution of monoamine oxidase and glucose-6-phosphatase activity, but the preparation was largely free of sarcoplasmic reticulum, mitochondrial inner, and lysosomal membranes.     

Purification of rabbit lacrimal gland plasma membranes by aqueous two-phase affinity partitioning

We describe the purification of lacrimal gland plasma membranes by affinity partitioning using a two-phase system containing polyethylene glycol and dextran in which wheat germ agglutinin conjugated to dextran is used as affinity ligand. When partitioning a microsomal fraction, the plasma membrane marker 5′-nucleotidase was obtained in the affinity ligand-containing bottom phase, whereas the endoplasmic reticulum marker NADH-ferricyanide reductase remained in the top phase. The affinity partitioning behaviour of components involved in exocytosis and cellular signalling was also examined.     

Spectroscopic and calorimetric studies on the interaction of human serum albumin with DPPC/PEG:2000-DPPE membranes

Site specific spectroscopic techniques and differential scanning calorimetry were used to study human serum albumin (HSA) in the absence and in the presence of membranes composed of dipalmitoylphosphatidylcholine (DPPC) and poly(ethylene glycol:2000)-dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE). Electron spin resonance (ESR) of a maleimide spin-label (5-MSL) covalently bound to the free sulfhydryl group at the unique cystein Cys-34 in domain I, intrinsic fluorescence of the single tryptophan Trp-214 in domain II, and extrinsic fluorescence of p-nitrophenyl anthranilate conjugated with tyrosine Tyr-411 in domain III were employed to study HSA dispersions with or without polymer-grafted membranes. On adsorbing at the DPPC membrane surfaces, domain I assumes a more loosened conformation and partitioning of the spin-labelled protein between the aqueous phase and the interfacial region of lipid membranes is observed by ESR. Domain II and III undergo a local structural arrangement which leads Trp-214 and Tyr-411 to come closer and causes intrinsic fluorescence quenching. The influence of DPPC bilayers on HSA is characterized both by a decrease of the thermal unfolding enthalpy and by a slight increase of the transition temperature, T (t), of the protein. The lipid induced effects on HSA are progressively reduced on increasing the amounts of PEG:2000-DPPE mixed with DPPC from the mushroom regime to the brush regime. Primary protein adsorption at the lipid surfaces is abolished at 1 mol% of the polymer-lipid, whereas the secondary protein adsorption at the polymer-brush leads to a further increase of both transition enthalpy and T (t) relative to the case of aqueous dispersions of HSA alone.     

Adsorptive membranes for bilirubin removal

In this study, we employed ethylene vinyl alcohol (EVAL) adsorptive membranes with bovine serum albumin (BSA) as bioligand for affinity supports for bilirubin (BR) retention. Microfiltration membranes were prepared from ternary or quaternary water/(1-octanol)/DMSO/EVAL systems. To obtain active binding sites for BSA, the EVAL membranes were either chemically functionalized in aqueous and organic medium and by plasma dischargement or physically activated by entrapping of active particles. Static BR removal was determined for all EVAL-BSA membranes. BR retentions relevant for human plasma were gained for the mixed adsorber membranes and additionally investigated in the dynamic mode.