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At least 19 recordsLinked to original sources

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed1998

[Study on immobilized cells for producing alpha-amylase by using polyving alcohol as the carrier(II): The effect of fermentating conditions on the ability producing alpha-amylase of the cells immobilized with polyving alcohol as the corrier and continuous fermentation of the immobilized cells in CSTR].

The effects of fermentating conditions on the ability of immobilized cells with PVA as carrier for producing alpha-amylase were studied. The continuous fermentation with the immobilized cells were tested in continuous flow stirred tank reactor (CSTR). The results showed that the adaptability of the immobilized Bacillus substilis to pH increased after immobilization. In CSTR, the immobilized cells can be fermentated continuously for 360 hrs and the activity of alpha-amylase can be kept on the level of about 170 u/ml.

Bacillus↗

PubMed1982

Steroid modifications with immobilized biocatalysts--use of immobilized enzyme-requiring cofactor regeneration and of immobilized mycelium.

Two biological approaches have been investigated for specific modifications of steroids. The first one uses the purified enzyme for the specific dehydrogenation of androsterone to androstanedione. The enzyme used is 3 alpha-hydroxysteroid dehydrogenase which requires a cofactor (NAD). A cofactor regeneration is needed so that the process could work continuously. The conjugation of two points (immobilization of the enzyme and optimization of the ratio methanol-water) allows a continuous work of the enzyme during 25 days. Moreover, we propose a chemical regeneration of the cofactor using methoxy derivative of phenazine methosulphate. Right now it is the limiting step of the process. The second approach of steroid modification uses a whole mycelium of Aspergillus phoenicis for the specific hydroxylation of progesterone to 11 alpha-hydroxy progesterone. The transformation of 90% of the progesterone is obtained with calcium alginate immobilization and the lowest number of products is obtained at pH lower than 2.5 with carrageenan and polyurethane immobilization. It seems promising to apply immobilized biocatalysts to the bioconversion of hydrophobic compounds in organic solvents system.

3-Hydroxysteroid Dehydrogenases↗

PubMed1983

Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions.

Immobilized metal ion affinity adsorption (IMA adsorption) is a collective term that is proposed to include all kinds of adsorptions whereby metal atoms or ions immobilized on a polymer cause or dominate the interaction at the sorption sites. IMA chromatography is one of the most powerful methods available to date for protein fractionation although this is not as yet widely recognized. This study deals with the theoretical aspects of IMA adsorption and its practical applications as exemplified by the various results reported here. The synthesis of iminodiacetate-substituted agarose (IDA-agarose) and tris(carboxymethyl)ethylenediamine-agarose (TED-agarose) is described. Many types of metal ions can easily be immobilized on these gel derivatives to form IMA adsorbents. We have not observed any damage to the proteins during the adsorption-desorption process. After performance of an experiment, the gels can easily be regenerated and can be loaded with the same or a different metal ion for an ensuing experiment. Specific adsorption is demonstrated for serum proteins on immobilized Ni(II) and Fe(III). Ligand-specific desorption (affinity elution) is also demonstrated by including in the buffer system certain solutes which are similar to or identical with some particular amino acids found in proteins. High concentrations of certain salts that affect the structure of water, such as Na2SO4, promote coordinate covalent bonding of proteins by a mechanism that is apparently similar to that found in hydrophobic interactions. Neutral detergents and aquoorganic solvents may be used. This opens up the possibility for the fractionation of membrane components. The IMA-adsorption method could also be expanded to other areas besides protein fractionation.

Adsorption↗

PubMed1999

Immobilization patterns and dynamics of acetate-utilizing methanogens immobilized in sterile granular sludge in upflow anaerobic sludge blanket reactors.

Sterile granular sludge was inoculated with either Methanosarcina mazeii S-6, Methanosaeta concilii GP-6, or both species in acetate-fed upflow anaerobic sludge blanket (UASB) reactors to investigate the immobilization patterns and dynamics of aceticlastic methanogens in granular sludge. After several months of reactor operation, the methanogens were immobilized, either separately or together. The fastest immobilization was observed in the reactor containing M. mazeii S-6. The highest effluent concentration of acetate was observed in the reactor with only M. mazeii S-6 immobilized, while the lowest effluent concentration of acetate was observed in the reactor where both types of methanogens were immobilized together. No changes were observed in the kinetic parameters (Ks and mumax) of immobilized M. concilii GP-6 or M. mazeii S-6 compared with suspended cultures, indicating that immobilization does not affect the growth kinetics of these methanogens. An enzyme-linked immunosorbent assay using polyclonal antibodies against either M. concilii GP-6 or M. mazeii S-6 showed significant variations in the two methanogenic populations in the different reactors. Polyclonal antibodies were further used to study the spatial distribution of the two methanogens. M. concilii GP-6 was immobilized only on existing support material without any specific pattern. M. mazeii S-6, however, showed a different immobilization pattern: large clumps were formed when the concentration of acetate was high, but where the acetate concentration was low this strain was immobilized on support material as single cells or small clumps. The data clearly show that the two aceticlastic methanogens immobilize differently in UASB systems, depending on the conditions found throughout the UASB reactor.

Acetates↗

PubMed2001

Optimization of the immobilization parameters and operational stability of immobilized hydantoinase and l-N-carbamoylase from Arthrobacter aurescens for the production of optically pure l-amino acids.

2The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His(6)-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH(2))-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements.In comparison to the free enzymes, temperature and pH-optima were increased by 10 degrees C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60 degrees C (60 degrees C).In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.

Journal Article↗

PubMed1988

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents.

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.

Chromatography, Affinity↗

PubMed1985

Changes in the colonic temperature and metabolism during immobilization stress in repetitively immobilized or cold-acclimated rats.

Effect of immobilization stress on the rat colonic temperature and metabolism was studied in a warm environment of 25 degrees C. Immobilization for 3 hr caused hyperthermia accompanied by increased oxygen consumption (VO2) in the warm controls. The hyperthermic effect of immobilization was accelerated after 1 week repetition of daily immobilization and it was lessened after 2 to 4 week repetition of daily immobilization. The magnitude of VO2 increase was the same throughout the experimental period of 4 weeks during the immobilization. Hypothermia was never observed during immobilization. Immobilization-induced hyperthermia was significantly potentiated in the cold-acclimated rats, while VO2 increase did not differ between cold-acclimated rats and warm controls. These results suggest that immobilization stress causes the changes in body temperature through thermogenic and heat-loss mechanisms.

Analysis of Variance↗

PubMed1993

Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme.

Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.

Biotechnology↗

PubMed1980

[Immobilized luciferase from the fireflies Luciola mingrelica. Changes in the pH-dependence of the enzyme catalytic activity and stability during its immobilization on various polysaccharide carriers].

The luciferase from the fireflies luciola mingrelica was immobilized on different CNBr-activated polysaccharide carriers, e. g. Sepharose, Ultrodex, cellophane, Ultrogel. The catalytic activity and the inactivation rate constants of the soluble and immobilized enzyme at 25 degrees (pH 6.0--9.0) were determined. During immobilization on Sepharose the pH profile of activity shifts towards lower pH values, while upon immobilization on Ultrogel and Ultrodex it is considerably broadened. Immobilization on Ultrogel and Ultrodex results in a 3--100 -fold stabilization of the enzyme (pH less than 7.5). whereas the stability of luciferase immobilized on Sepharose and cellophane within the same pH range is 10--1000 times higher than that of the soluble enzyme. It was shown that inactivation of luciferase immobilized on Sepharose and cellophane is limited by oxidation of the SH-groups of the enzyme and is inhibited by dithiothreitol. The inactivation of luciferase immobilized on Ultrogel and Ultrodex and that of soluble enzyme is not limited by oxidation of the SH-groups.

Animals↗

PubMedDate not supplied

Immobilization of human thrombomodulin onto biomaterials. Comparison of immobilization methods and evaluation of antithrombogenicity.

Human thrombomodulin (hTM), which is a newly described endothelial cell associated protein that functions as a potent natural anticoagulant by converting thrombin from a procoagulant protease to an anticoagulant, was immobilized on to various substrates by two immobilization methods. As the substrates of immobilization, poly(acrylic acid) surface grafted poly(ethylene) (PAAc-g-PE) film, poly(vinylamine) surface grafted poly(ethylene) film, and PAAc surface grafted nylon were used. For immobilization, simultaneous preactivation methods were used. The effect of the immobilization reaction on hTM activities, the comparison of the activities of immobilized hTM with those of free hTM, and the effect of the thrombin incorporation on antithrombogenic activity were studied. The hTM immobilized onto PAAc-g-PE by preactivation showed the highest antithrombogenic activity. The thrombin incorporation affected protein C activation activity but not the fibrinogen clotting time. hTM immobilized nylon showed greater antithrombogenicity in vitro.

Anticoagulants↗

PubMed1981

Immobilization osteoporosis and active vitamin D: effect of active vitamin D analogs on the development of immobilization osteoporosis in rats.

The therapeutic effects of vitamin D analogs, 1,24(R)-dihydroxycholecalciferol [1,24(R)-(OH)2D3], 1,24(S)-dihydroxycholecalciferol [1,24-(S)(OH)2D3], and 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on immobilization osteoporosis were studied in rats. The right hind limb was immobilized through application of a plaster cast following the section of the sciatic nerve. The left hind limb was intact. Vitamin D analogs were orally administered for 6 weeks at dose levels of 0.02 and 0.10 micrograms/kg/day, respectively. The mean lengths of the immobilized femurs were not significantly different from those of the intact femurs in all the experimental groups. In the immobilized femur of animals treated with 1,24(R)(OH)2D3, 0.10 micrograms/kg, dry and ash weights were heavier and calcium and phosphorus contents greater than those in the nontreated group. Furthermore, the amount of calcified bone mass and the cortical thickness of the femurs of the immobilized limb in 1,24(OH)2D3-treated animals were greater than those in the nontreated animals. Treatment with 1,25(OH)2D3 at 0.10 micrograms/kg caused an increase of the bone mass in both immobilized and intact femurs when compared with those of the control group. It was concluded that the administration of 1,24(R)(OH)2D3 diminished the effect of immobilization in the development of osteoporosis without any side effects.

Animals↗

PubMedDate not supplied

A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of beta-galactosidase from Aspergillus oryzae.

The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the beta-galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).

Amino Acids↗

PubMed1978

Coupled reactions of immobilized enzymes and immobilized substrates: clinical application as exemplified by amylase assay.

We described a partitioned enzyme-sensor system, which incorporates an immoblized substrate and three or more discrete immobilized enzymes. This instrument measures alpha-amylase activity by passing the solution containing alpha-amylase over a column packed with immobilized starch. The resulting oligosaccharides are successively exposed to a column or columns containing immobolized glucose oxidase, catalase, glucoamylase or maltase, and glucose oxidase. The resulting hydrogen peroxide is detected by a three-electrode amperometric cell. All immobilized reagents were immobilized on a particulate, porous alumina to allow rapid and constant flow rate. With use of less than optimum immobilized reagents, alpha-amylase activity has been measured from about 5 to 200 kU/liter with a 50 microliter sample size. Lack of sensitivity is predominantly attributable to the low activity and low stability of immobilized maltase and glucoamylase. We believe that a clinical test using this system is feasible and desirable because the immobilized reagent system should allow for testing of alpha-amylase with excellent precision, convenience to the operator, and low cost.

Amylases↗

PubMed1982

[Use of immobilization in the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized monomers].

Active immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase were prepared by means of dissociation of the tetrameric enzyme molecule covalently bound to Sepharose via a single subunit. The conditions were elaborated to achieve the inactivation and solubilization of the non-covalently bound subunits leaving the monomer coupled to the matrix intact. This procedure differs from the previously developed method of matrix-bound oligomeric enzymes dissociation in a detail which was found to be essentially important. The widely used method includes complete denaturation of all subunits during treatment with urea followed by reactivation of the immobilized one, whereas only the non-covalently bound subunits suffer denaturation under the conditions developed in the present work. The immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase exhibit Vmax and Km (for NAD and substrate) values similar to those found for the immobilized tetramer. Reassociation of the immobilized monomers with soluble enzyme subunits obtained in the presence of urea produces matrix-bound tetrameric species. Immobilized trimers ae formed upon incubation of matrix-bound monomers in a diluted apoenzyme solution. The immobilized monomeric, trimeric and tetrameric enzyme species were used to study the role of subunit interactions in cooperative phenomena exhibited by the dehydrogenase.

Enzymes, Immobilized↗

PubMedDate not supplied

[Immobilization of enzymes on carriers with magnetic properties: the search for optimum conditions for immobilization of alkaline phosphatase from the chicken gut].

Chicken intestine alkaline phosphatase was immobilized on seven carriers by adsorption or covalent binding. The efficiency of immobilization depended on the carrier, immobilization method, and initial enzyme concentration. The heat stability of the soluble and immobilized enzyme preparations, and the dependence of the enzyme activity on pH and Zn2+ and Mg2+ ions were studied. Alkaline phosphatase immobilized on magnetic carriers displayed lower heat stability than the soluble enzyme. The temperature optima of the immobilized and soluble enzymes were 80 and 60 degrees C; the pH optima were 9.0 and 8.0, respectively. Ions Mg2+ and Zn2+ were found to play an important role in the activation of both soluble and immobilized enzymes and their stabilization at high temperatures.

Alkaline Phosphatase↗

PubMed1992

Cell growth on immobilized cell-growth factor; 4: Interaction of fibroblast cells with insulin immobilized on poly(methyl methacrylate) membrane.

Insulin was immobilized on the surface-hydrolyzed poly(methyl methacrylate) membrane and the growth acceleration of mouse fibroblast cells, STO, by the immobilized insulin was investigated. It was found that insulin remains immobilized on the surface of nonbiodegradable membrane and interacts specifically with receptors existing on the biological membrane of fibroblast cells. The growth acceleration by immobilized insulin was enhanced by introduction of a spacer arm between insulin and the immobilization matrix. The amount of receptor proteins present on the biological membrane of fibroblast cells after culturing with insulin, immobilized on nonbiodegradable polymer membrane, was much higher than that after culturing with free insulin, implying the suppression of down-regulation in the case of immobilized insulin.

Animals↗

PubMed2003

Syntheses of immobilized G protein-coupled receptor chromatographic stationary phases: characterization of immobilized mu and kappa opioid receptors.

Opioid receptors are members of the superfamily of G protein-coupled receptors (GPCRs). Liquid chromatographic stationary phases containing either the human mu or kappa opioid receptor subtypes have been developed to study the binding between the opioid receptors and their ligands. The receptors were obtained from Chinese hamster ovary cells stably transfected with human mu or kappa receptor subtypes. The receptors were isolated through partial solubilization with sodium cholate detergent, and the partially purified receptor complex was immobilized in the phospholipid analogue monolayer of an immobilized artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in the on-line chromatographic determination of drug/ligand binding affinities to the immobilized opioid receptors. Preliminary on-line binding studies employing frontal chromatographic techniques were conducted with the known mu antagonist (naloxone) and a kappa agonist (U69593). The calculated dissociation constants (Kd) were 110 nM for naloxone and 84 nM for U69593. The results indicate that the immobilized receptors retained their ability to specifically bind ligands and were active for 1200 column volumes applied over two weeks. Zonal chromatographic experiments were also conducted, and competitive displacements of the marker ligands were observed. The results suggest that the immobilized opioid receptor stationary phases can be used to qualitatively assess the binding affinities of compounds to the immobilized receptors and represent the first example of the use of immobilized GPCRs in a chromatographic system.

Animals↗