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Biomedical subjects

H Umakoshi

Publications and source records attributed to H Umakoshi.

15 recordsLinked to original sources
PubMed2000

Immobilized liposome chromatography for refolding and purification of protein.

Small unilamellar liposomes were utilized as a kind of aqueous two-phase system and artificial chaperone which specifically recognize protein conformation with fluctuated structure. Liposomes showed highly selective binding ability to conformationally changed proteins treated with various concentrations of guanidinium hydrochloride, as evaluated by immobilized liposome chromatography (ILC). In refolding of proteins, liposomes bound to refolding intermediate of proteins and prevented them from forming intermolecular aggregates. Refolding of bovine carbonic anhydrase, lysozyme and ribonuclease A was significantly improved in the presence of liposomes. Furthermore, by utilizing ILC, refolding of proteins was also successfully and simply carried out with considerable high reactivation yield.

Animals↗
PubMed2000

Stimuli-responsive separation of proteins using immobilized liposome chromatography.

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k(s)) of beta-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k(s),max = 5.57). The k(s) values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k(s), therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (alpha-glucosidase, k(s),max = 11.3 at pH 4; carbonic anhydrase from bovine, k(s),max = 6.53 at pH 4). The difference in the height and/or the position of the peaks of the ks-pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.

Animals↗
PubMed2000

Protein refolding using stimuli-responsive polymer-modified aqueous two-phase systems.

The function of a stimuli-responsive polymer was studied for the utilization of protein unfolding and refolding in protein separation using aqueous two-phase systems (ATPS). Poly(ethylene glycol) (PEG) bound to a thermo-reactive hydrophobic head (poly(propylene oxide)-phenyl group (PPO-Ph group)) was used as the functional ligand to modify the PEG phase of the aqueous two-phase systems. Firstly, refolding of carbonic anhydrase from bovine (CAB) was examined in the presence of PPO-Ph-PEG at various temperatures. The refolding yield of CAB was strongly enhanced and aggregate formation was suppressed by addition of PPO-Ph-PEG at a specific temperature (50-55 degrees C). The change in the local hydrophobicity of CAB and PPO-Ph-PEG was characterized using the aqueous two-phase partitioning method and a hydrophobic fluorescent probe. The local hydrophobicity of CAB was maximized at 60 degrees C. The local hydrophobicity of PPO-Ph-PEO was also found to be increased above 45 degrees C. A simple model for CAB refolding, which includes (i) PPO-Ph-PEG complex formation and CAB in the intermediate state and (ii) refolding and release of native CAB from the PPO-Ph-PEG surface, is suggested based on the evaluated surface hydrophobicity.

Animals↗
PubMed2000

Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems.

For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 50 degrees C, the partition coefficients of CI2 in Breox-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50 degrees C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60 degrees C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox-dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.

Chymotrypsin↗
PubMed2000

Dielectric response of cells and liposomes and its utilization for evaluation of cell membrane-protein interaction.

The dielectric measurements of an Escherichia coli (E. coli) cell suspension and liposome suspensions were carried out in the frequency range between 0.1 and 100 MHz to detect the heat stress-mediated interaction between proteins and cell membranes. The dielectric relaxation dispersion was observed to be above 1 MHz. The dielectric parameter (amplitude of dispersion, deltaepsilon) based on the Cole-Cole equation was anomalously changed with increasing temperature. The value of deltaepsilon of liposomes was varied at various temperatures depending on the type of protein present. The change in deltaepsilon of liposomes correlated with the amount of protein translocated across the phospholipid membrane. The changes in the value of deltaepsilon of E. coli cells with temperature variation was similar to those of liposomes in the presence of proteins, suggesting that the variation in dielectric parameters reflected the interaction between the phospholipid membrane and proteins. It was found that the dielectric measurement could be utilized for the detection of the interaction between proteins and liposomes.

Journal Article↗
PubMed1999

Characterization and on-line monitoring of cell disruption and lysis using dielectric measurement.

The dielectric measurement of Escherichia coli suspension was carried out between 0.01 and 100 MHz during cell disruption using a physical method or cell lysis induced by chemicals for the purpose of on-line monitoring of cell disruption or lysis of bacterial cells. The dielectric dispersion of relative permittivity centered at about 1.4 MHz and the dielectric parameters based on the fitting calculation well reflected the damage of these cells under physical and chemical stresses. The degree of cell disruption as determined by the dielectric parameters, the amplitude of dispersion and the conductivity, well corresponded to those obtained by other conventional kinetic analyses. This methodology can be utilized for the on-line monitoring of physical cell disruption and cell lysis induced by detergent and could provide direct information to control these processes. Based on these results, the dielectric measurement was successfully applied to monitor the stress-mediated cultivation of E. coli cells.

Journal Article↗
PubMed1998

Selective separation process of proteins based on the heat stress-induced translocation across phospholipid membranes.

Heating of several protein solutions at 40-47 degrees C for 5-60 min in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes induced the translocation of beta-galactosidase (beta-gal), alpha-glucosidase (alpha-glu) and bovine carbonic anhydrase (CAB) from outer to inner aqueous phase across the liposome membrane. The translocated amounts of beta-gal at various temperatures were maximized under suitable heating conditions (45 degrees C, 30 min). Those of alpha-glu and CAB were maximized at 40-45 and 60 degrees C, respectively. Each maximum value could be correlated with the corresponding local hydrophobicity of each protein evaluated by the aqueous two-phase partitioning method. The possibility to apply these heat-induced translocation phenomena to the bioseparation of proteins was successfully demonstrated for the model mixture solution of beta-gal, alpha-glu and CAB.

Biological Transport↗
PubMed1998

Enzyme release from heat-stressed cell membranes as a function of hydrophobicity evaluated by using aqueous two-phase systems.

The release behavior of a periplasmic enzyme, acid phosphatase, from heat-stressed Escherichia coli cells was characterized by using kinetic analyses when the cells were treated by Triton X-100-EDTA. The hydrophobicity of the cell surface and the release-rate of the enzyme were not influenced by heat treatment at temperatures between 30 and 50 degrees C. However, these values varied above 55 degrees C. The release-rate constants were found to correspond to the net and local hydrophobicity of the outer membrane surface, evaluated by aqueous two-phase partitioning.

Acid Phosphatase↗
PubMedDate not supplied

Extractive cultivation of Escherichia coli using poly(ethylene glycol)/phosphate aqueous two-phase systems to produce intracellular beta-galactosidase.

Escherichia coli cells were found to grow in poly(ethylene glycol) (PEG)/phosphate aqueous two-phase systems by selecting the phase-forming components and their concentrations, the tie-line length, and the phase volume ratio properly. The cells cultivated up to an optical density at 660 nm of 1.0 were disrupted by ultrasonic irradiation to release and recover overproduced beta-galactosidase. The surviving cells were found to grow immediately after ultrasonic irradiation. The PEG/phosphate (KH2-PO4-K2HPO4) system with added Na2SO4 was the one optimized for extractive cultivation of E. coli cells, where beta-galactosidase was selectively partitioned to the top phase while total soluble proteins and cells partitioned to the bottom phase. This integrated process was extended to a semicontinuous operating mode, where the top phase containing beta-galactosidase was removed following intermittent ultrasonic irradiation and the bottom phase containing cells was recycled together with the new top phase solution to repeat production and recovery of beta-galactosidase.

Cell Division↗
PubMedDate not supplied

Extractive cultivation of recombinant Escherichia coli using aqueous two-phase systems for production and separation of intracellular heat shock proteins.

The extractive cultivation of recombinant Escherichia coli cells to produce, release, and separate heat shock proteins (HSPs; GroEL and GroES) using poly(ethylene glycol) (PEG)/dextran (Dex) aqueous two-phase systems was developed. The growth rate of E. coli OW10/pND5 cells in the PEG/Dex two-phase media was almost the same value as that in the control media. The addition of 0.1 M potassium phosphate salts (KPi) increased the productivity of HSPs with keeping the growth rate of E. coli cells relatively high. The partition coefficients of HSPs were improved to greater values when phosphate salts were added at a concentration of more than 0.1 M. As a result, PEG/Dex systems supplemented with 0.1 M KPi were found to be the optimized two-phase systems for the extractive cultivation of E. coli cells. In the systems, the HSPs were selectively partitioned to the top phase while cells occupied the bottom phase and the interface between the two phases. This integrated process was extended to a semicontinuous operating mode, where the top phase containing the HSPs was recovered following intermittent heating and ultrasonic irradiation. The bottom phase containing cells and cell debris was recycled together with new top phase solution to repeat production and recovery of HSPs.

Culture Media↗
PubMedDate not supplied

Heat-induced translocation of cytoplasmic beta-galactosidase across inner membrane of Escherichia coli.

The behaviors of heat-induced translocation of cytoplasmic beta-galactosidase to periplasm across the inner membrane of Escherichia coli cells were investigated in order to apply such phenomena to the process for production and separation of intracellular biomolecules. The heat stress was found to induce translocation of cytoplasmic beta-galactosidase (beta-gal) together with reduction of the amounts of intracellular soluble proteins and formation of their inactive aggregates. The translocation of beta-gal was then analyzed using (a) the location factor of beta-gal (LFG), which meant enzyme location in the cells and could be determined from the kinetic analysis of enzyme release process, and (b) the percentage of beta-gal activity in periplasm after solublizing the outer membrane of E. coli cells by lysozyme/EDTA treatment. The LFG values were maximized when cells were stressed at the temperature of 42-47 degrees C. From the results on the surface properties of both beta-gal and cell membrane under the heat stress, it is suggested that (1) the conformational change of cytoplasmic oligomeric beta-gal to the partially dissociated and/or unfolded state with higher local hydrophobicity, (2) the increase in membrane fluidity of inner membrane, (3) the enhancement of hydrophobic interaction between lipid and protein, and (4) the inhibition of its translocation by GroEL restabilizing the proteins could underlie the heat-induced translocation of beta-gal across the inner membrane. The possibility to apply the heat-induced translocation of beta-gal for the enhancement of the target selectivity at the process upstream is finally presented.

Bacterial Proteins↗
PubMedDate not supplied

Model system for heat-induced translocation of cytoplasmic beta-galactosidase across phospholipid bilayer membrane.

The possibility of the translocation of the enzyme across the phospholipid bilayer membrane was investigated by using the liposomes prepared by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in which beta-galactosidase (beta-gal) was entrapped. Exposing the POPC liposomes entrapping beta-gal inside to heat treatment (40-50 degrees C, 1-60 min) was found to induce its translocation across the liposome membrane. The translocated activity of beta-gal from inner to outer aqueous phase of liposomes indicated the maximal value when the liposomes entrapping beta-gal were heated at 45 degrees C for 30 min. The gel permeation profiles of the liposomes before and after heat treatment (45 degrees C, 30 min) also supported the translocation of beta-gal across the liposome membrane. The membrane fluidity of liposomes was found to be increased with increasing temperature, so that the hydrophobicity of liposome membrane was also increased. The local hydrophobicity of beta-gal was maximized at the temperature of 40-50 degrees C. The mechanisms of beta-gal translocation have been suggested to be triggered by the enhancement of hydrophobic interaction between the liposome surface and beta-gal molecules. Finally, a minimal scheme of possible mechanism on the heat-induced translocation of beta-gal has been presented on the basis of the hydrophobic interaction between the liposome and the proteins. The experimental data on the heat-induced translocation of beta-gal were well corresponding to those from model calculation.

Chromatography, Gel↗
PubMedDate not supplied

Utilization of cell response under heat, chemical, and combined stresses for selective recovery of cytoplasmic beta-galactosidase from Escherichia coli cells.

The effective bioseparation process was developed by exploiting the cell response under heat, chemical, and combined stresses for selective recovery of cytoplasmic beta-galactosidase from Escherichia coli cells. At the observed optimal condition for heat stress (45 degrees C, 30 min) and that for chemical stress (20 mM Triton X-100, 5 mM EDTA, 30 min), E. coli cells were exposed to these stresses either simultaneously or sequentially. The operational sequence highly affected the yield and selectivity of the target recovery. Exposing the cells to the sequential stress (heating at 45 degrees C for more than 30 min after Triton X-100/EDTA treatment for 30 min) was found to be most effective for selective recovery of target beta-galactosidase among possible sequences.

Biotechnology↗
PubMedDate not supplied

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.

Cell Membrane Permeability↗