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PubMed2000

Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin.

A novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. The mcl-PHA polymerase 1 from Pseudomonas oleovorans was overproduced from the Palk promoter. Most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. By incubating the protein aggregates with S-Sepharose ion-exchange resin in the presence of dithiothreitol and glycerol, the mcl-PHA polymerase could be extracted in an active and soluble form with a final yield of about 5.2 mg/g of cell dry weight. The solubilized polymerase was able to catalyse the in vitro synthesis of mcl-PHA without any additional cell components, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activating purified inactive labile enzymes.

Acyltransferases↗
PubMedDate not supplied

The use of ion-exchange resins as a blood preservation system.

A system using ion-exchange resins for the storage of whole blood was investigated. Blood was collected into CPD, thoroughly mixed and then divided into equal amounts. To one half of the split units, four grams of phosphate charged Amberlite IR-45 resin were added, while no additions were made to the other split units. Both aliquots were stored at 4 C, adhering to routine blood banking standards. The blood stored with the resin for up to 28 days maintained high levels of 2,3-DPG and adequate levels of ATP. This new resin system has several advantages over the present blood preservation technology. There is initial increase in pH that promotes greater glucose utilization. Inorganic phosphate is provided to maintain both ATP and 2,3-DPG levels. The resin buffers the blood and maintains a narrower pH range for red blood cell enzyme activity. The resin is totally inert, completely filterable and has the ability to adsorb bacteria from the suspending solution.

Adenosine Triphosphate↗
PubMed2006

Using ion-exchange resins to study soil response to experimental watershed acidification.

Ion-exchange resins (IER) offer alternative approaches to measuring ionic movement in soils that may have advantages over traditional approaches in some settings, but more information is needed to understand how IER compare with traditional methods of measurement in forested ecosystems. At the Bear Brook Watershed in Maine (BBWM), one of two paired, forested watersheds is treated bi-monthly with S and N (28.8 and 25.2kg ha(-1)yr(-1) of S and N, respectively). Both IER and ceramic cup tension lysimeters were used to study soil solution responses after approximately 11 years of treatment. Results from both methods showed treatments resulted in the mobilization of base cations and Al, and higher SO(4)-S and inorganic N in the treated watershed. Both methods indicated similar differences in results associated with forest type (hardwoods versus softwoods), a result of differences in litter quality and atmospheric aerosol interception capacity. The correlation between lysimeter and IER data for individual analytes varied greatly. Significant correlations were evident for Na (r=0.75), Al (r=0.65), Mn (r=0.61), Fe (r=0.57), Ca (r=0.49), K (r=0.41) and NO(3)-N (r=0.59). No correlation was evident between IER and soil solution data for NH(4)-N and Pb. Both IER and soil solution techniques suggested similar interpretations of biogeochemical behavior in the watershed.

Ion Exchange Resins↗
PubMed1994

[An in vivo experiment on the antitumor action of bleomycin bound to an ion exchange carrier].

Bleomycetin, an antitumor antibiotic of the domestic origin, is component A5 of the bleomycin complex. Design of new drug delivery systems especially for local application is an important problem of antitumor chemotherapy. Bleomycetin immobilized on ion exchange medical gauze was studied with this purpose in an in vivo experimental model of murine lympholeukemia NK/Ly. Subcutaneous implantation of the new dosage form of bleomycetin with prolonged action provided a significant antitumor systemic effect evident from an increase in the mouse life-span. In vitro determination of the bleomycetin contents in the gauze revealed that the antibiotic was completely absorbed from the carrier within 72 hours. The bleomycetin half-life in the plasma after the antibiotic administration in the bound form on the ion exchange gauze was twice as long as that after the antibiotic injection with NaCl isotonic solution.

Animals↗
PubMed2000

Assay of Adenylyl Cyclase Activity by Ion-exchange High-performance Liquid Chromatography.

Adenylyl cyclase(AC)activity was determined by directly quantifying the product cAMP with ion-exchange HPLC. When AC reaction was terminated, the reaction mixture was kept at 0 degrees for 2 h to remove residual ATP utilizing the action of ATP-hydrolyzing enzymes existed in the crude AC preparation of membrane pellet. Papaverine was removed by the dichloromethane extraction, and cAMP was quantified by chromatography on a WAX-1 HPLC column with baseline-resolution. It was found that cAMP was linearly formed within 10 min of the reaction, and the amount of cAMP formed was proportional to the amount of enzyme. Mouse brain AC activity determined by this method was 42 nmol/min per gram of protein, similar to that obtained by the classical [(3)H] labeled ATP method. This ion-exchange HPLC was more rapid and convenient.

Journal Article↗
PubMed1984

Ion-exchange properties of Cibacron Blue 3G-A Sepharose (Blue Sepharose) and the interaction of proteins with Cibacron Blue 3G-A.

The affinity for Blue Sepharose of several proteins of known structure showed a pH dependence governed by their isoelectric points; Blue Sepharose behaved like a strong cationic ion exchanger because of the negative charges of its dye ligand, Cibacron Blue. A study of the protein-Cibacron Blue interactions by phase partition and equilibrium dialysis revealed the presence of high-affinity binding sites both in the case of the (di)nucleotide-dependent enzymes that possess the structural domain known as "dinucleotide fold", and in the case of other proteins consisting almost entirely of alpha-helix (human haemoglobin, cytochrome c) or beta-sheet (human immunoglobulin G). The presence of additional sites of low affinity, probably situated at the protein surface, was also inferred from the equilibrium dialysis data. In some instances, in contrast with the Sepharose-immobilized dye, the interaction of free Cibacron Blue with proteins was not pH dependent. Steric factors could be responsible for such a differential behaviour. It is suggested that certain nucleotide-dependent enzymes might also bind to Blue Sepharose by ion exchange. Preparative applications of these findings are illustrated and discussed in terms of the optimization of affinity chromatography experiments.

Animals↗
PubMed1994

Ion exchange resins for the purification of monoclonal antibodies from animal cell culture.

We have compared various ion exchangers for monoclonal antibody (MAb) purification using different starting materials such as ascitic fluid and cell culture supernatant. Twelve cation and anion exchange resins were tested so far. Purification of MAbs with regard to the starting material is described. In well-defined conditions of adsorption (20 mM MES buffer, pH 6.50), one purification step based on cation-exchange chromatography is generally sufficient to achieve at least 90% purity of the MAb, even when produced by animal cell culture. Cation-exchange supports exhibit higher capacity for MAbs compared to anion exchangers. Among the cation exchangers tested, we have selected the cross-linked matrix S Sepharose FF for its large specificity and capacity for MAbs. Considering these key parameters and also the good mechanical resistance of the S Sepharose FF, we describe how, by varying the flow rate, sample concentration, and size of the column, the productivity may be improved in a monoclonal antibody purification process. Finally, a general 'gram scale' purification protocol of MAbs produced by animal cell cultures is proposed. This protocol, based on economical adsorption conditions and three steps of elution (100 mM, 200 mM and 1 M NaCl), allows the recovery of highly purified MAbs.

Animals↗
PubMed1998

Capture of human monoclonal antibodies from cell culture supernatant by ion exchange media exhibiting high charge density.

A shortcut purification sequence for therapeutic proteins should consist of three steps: capture, purification, and polishing. Special emphasis has been put on direct capture of human monoclonal antibodies from culture supernatants with ion-exchangers avoiding pretreatment steps such as desalting, dilution, and other means to reduce the ionic strength. CM-HyperD, a cation-exchanger composed of an inorganic macroporous support filled with a viscoelastic gel with a high charge density was used. Capture of monoclonal antibodies from clarified hybridoma cell culture grown in media supplemented with fetal calf serum was investigated. Screening of different pH conditions and buffers for the load step showed that monoclonal antibodies were efficiently bound by CM-HyperD at pH 4.0 and 5.0 at an ionic strength equivalent to culture supernatant. Combination of negative purification with Q-Sepharose FF and capturing with CM-HyperD gave sufficient yield and resolution. Implementation of wash steps with higher conductivity did not improve the purity, but decreased the yield. Interestingly, high flow rates improved the purity. When antibodies were captured from serumfree culture supernatant the antibody could be eluted in a single peak with substantial reduction of contaminants. Capturing of antibodies by ion-exchange sorbents from culture supernatant is possible despite the high salt content.

Antibodies, Monoclonal↗
PubMed1998

Purification of tubulin and microtubule-associated proteins by membrane ion-exchange chromatography.

Microtubules, composed of tubulin and microtubule-associated proteins (MAPs), can be isolated using routine procedures from homogenates of vertebrate brain. Often, it is necessary then to purify the tubulin from the MAPs, and normally this purification is effected by standard techniques of ion-exchange chromatography. However, such procedures can be expensive, both in the consumption of buffers and other expensive components (e. g. GTP) and in investigator time. Here, we demonstrate that membrane ion exchangers mounted in syringe filter cartridges can be used to separate tubulin from MAPs in a matter of minutes, compared to the several hours that are normally required for typical chromatographic procedures using phosphocellulose orDEAE. The resulting tubulin is competent to assemble into microtubules upon either addition of the purified MAPs or addition of the microtubule-stabilizing drug Taxol. Thus, the procedure should be useful to investigators requiring a rapid and effective purification of tubulin for use in assembly studies or in vitro motility assays.

Animals↗
PubMed2004

A pilot study on phosphate and nitrate removal from secondary wastewater effluent using a selective ion exchange process.

A pilot study was conducted at a wastewater treatment plant (WWTP) in Easton, Pennsylvania, for the removal of varying concentrations of phosphate (HPO4(2-)) and nitrate NO3- using a newly developed ion exchange material referred to as Polymeric Ligand Exchanger (PLE). The PLE was prepared by immobilizing copper (II) ions onto a commercially available chelating resin, DOW-3N. The loaded copper ions act as ligand exchanger sites that selectively bind with target ligands such as phosphate and nitrate. This pilot study was the final experimental testing phase of the PLE toward the full process development for the selective removal of both phosphate and nitrate using secondary effluent from a fully operational WWTP. In order to establish the effectiveness of the PLE, a commercially available activated alumina (AA) was also tested for comparison. Effluent phosphorus and nitrate concentration of less than 0.1 mg l(-1) were achieved for up to 500 bed volumes; phosphorous was reduced from an average concentration of 4.0 mg l(-1) and nitrate from average concentration of 16.4 mg l(-1). In accord with prior laboratory observations, the pilot test results showed that the PLE could selectively remove phosphate from secondary municipal wastewater effluent under normal conditions (pH, ionic strength, dissolved organic carbon content). In addition, the pilot study provided new evidence that nitrate could be removed from secondary municipal wastewater effluent and concurrently with phosphate. The selective ion exchange process provides an alternative for concurrent removal of the eutrophic-enhancing nutrients, phosphate and nitrate.

Copper↗
PubMed1999

Ion exchange chromatography of aluminum using 3-carboxy-2-naphthylamine-N,N-diacetic acid as a fluorescent post-column chelating reagent.

Ion exchange chromatography of aluminum ion using 3-carboxy-2-naphthylamine-N,N-diacetic acid (CNDA) as a fluorescent post-column chelating reagent was studied. The solution containing ammonium chloride and hydrochloric acid was used for the eluent, and acetate buffer solution containing CNDA was used for the post column chelating reagent. The peak of aluminum was separated from that of calcium, magnesium and zinc, and the chromatogram was not affected by copper(II) and iron(III). The calibration curve gave linear plots with a range of 0.0027-0.54 ppm aluminum, the regression coefficient of correlation (r2) was 1.000, and the detection limit (S/N = 3) was 0.3 ppb, indicating that the method could determine aluminum with high sensitivity. It was demonstrated that CNDA is a useful metallofluorescent reagent for aluminum. This method has been successfully applied to the determination of aluminum in some tea drinks.

Aluminum↗
PubMed1985

Application of a new ion exchanger TSK-GEL DEAE-5PW, to the purification of Cu,Zn-superoxide dismutase of bovine erythrocytes.

We have developed an effective method for the purification of Cu,Zn-super-oxide dismutase [E.C. 1.15.1.1] from bovine erythrocytes. This enzyme functions as a scavenger of superoxide radical, and it seems to be a key enzyme in the metabolism of active oxygen species. Application of this enzyme as a drug has been considered, and for this purpose a highly purified preparation is necessary. The first purification of this enzyme was reported by McCord and Fridovich [J. Biol. Chem., 244 (1969) 6049]. The limiting step in their method is the removal of the small amount of contaminating protein from the acetone-precipitated crude preparation (3000 units/mg protein). We found that a new, high-performance ion exchanger, TSK-GEL DEAE-5PW, improves this step and makes possible the large-scale preparation of pure protein. Optimal conditions for a TSK-GEL DEAE-5PW preparative column (150 X 21.5 mm I.D.) were established. The acetone-precipitated crude enzyme was dissolved in 20 mM Tris-HC1 buffer (pH 7.5), protein concentration 11.0 mg/ml, applied to the column, and eluted with a linear gradient of sodium chloride from 0 to 0.3 M. The flow-rate and sample volume were set to 4.0 ml/min and 8.0 ml, respectively. Under these conditions, the fractionated Cu,Zn-superoxide dismutase showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ion exchange high-performance liquid chromatography, and a specific activity of 3800 units/mg protein. The recovery of activity was 75%, and the cycle time was 120 min. A yield of 80-85 mg of purified enzyme was obtained per cycle. The preparation thus isolated has the highest purity and activity so far reported. We conclude that TSK-GEL DEAE-5PW is the practical choice for large-scale purification of Cu,Zn-superoxide dismutase.

Animals↗
PubMed1998

Sodium ion/hydrogen ion exchange inhibition: a new pharmacologic approach to myocardial ischemia and reperfusion injury.

Over the past few years, it has been shown that the cardiac myocyte plasma membrane sodium ion/hydrogen ion exchanger (NHE) plays an important role in the maintenance of intracellular pH, sodium, and calcium ion homeostasis. From the results of various experimental studies, it is clear that this ion exchanger is an important mediator of ischemic-reperfusion injury of the heart. During myocardial ischemia, intracellular acidosis develops quickly, activating the exchanger to extrude H+ into the extracellular environment and bring Na+ into the cell. With further progression of ischemia, the cell is unable to handle the overload of Na+, causing it to use its Na+/Ca2 exchanger to unload intracellular Na+ into the extracellular space. At the same time, however, calcium is being transported into the cell. This can lead to detrimental cardiac injury, such as contracture and necrosis. During myocardial reperfusion, these events are magnified because the return of blood flow lowers the extracellular H+ concentration, stimulating the NHE to extrude more intracellular H+ ion. This leads to intracellular Na+ excess and eventually, intracellular Ca2+ overload and cardiac injury. In an effort to alter these pathophysiologic events, a number of investigators have studied the ability of various NHE inhibitors, such as amiloride, analogues of amiloride, and other drugs (HOE 694, HOE 642), to prevent cardiac ischemic-reperfusion damage. Preliminary results from studies in animal models have revealed that most of these agents are able to attenuate the development of myocardial contracture, infarction, and arrhythmias during both ischemia and reperfusion. Their efficacy and cardioprotective effects in human beings have yet to be determined. These agents appear to be promising not only in the prevention and treatment of ischemic heart disease, but also in avoiding cardiac damage in situations where low-flow states are followed by immediate recovery of flow, as in coronary artery bypass graft surgery, percutaneous transluminal coronary angioplasty, thrombolytic therapy, and coronary arterial vasospasm. This article reviews the physiology of the NHE and analyzes the potential role of NHE inhibitors in the prevention of ischemic-reperfusion injury and other cardiac disease states.

Amiloride↗