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PubMed1992

Cell growth on immobilized cell growth factor. I. Acceleration of the growth of fibroblast cells on insulin-immobilized polymer matrix in culture medium without serum.

Culture of fibroblast cells in serum-free medium in the presence of insulin-immobilized films of poly(ethylene terephthalate) or poly(methyl methacrylate) accelerated the cell growth to several times that in the presence of free insulin molecules. The immobilization of insulin was useful also for elucidation of the mechanism of signal transmission through receptor molecules in the cell membrane. Free insulin molecules are known to be bound by receptors and the complex formed internalized and degraded in the cytoplasm. However, it is not clear what process is indispensable for transmission of the biological signals to the nucleus of the cell. The present investigation revealed that cell growth was enhanced by the addition of immobilized insulins which were not internalized. It appears that insulin-receptor binding is enough for transmission of the cell growth signal to the cell nucleus. The enhanced cell growth by the immobilized insulins compared with free insulin molecules is ascribable to suppression of the down-regulation which is a consequence of intracellular decomposition of the insulin-receptor complex.

Animals↗
PubMed1994

Cell growth on immobilized cell-growth factor. 10. Insulin and polyallylamine co-immobilized materials.

Polyallylamine, which is cationic and enhances cell adhesion electrostatically (non-specifically), was co-immobilized with a cell growth factor, insulin, onto a surface-hydrolysed poly(methyl methacrylate) membrane. The adhesion of mouse STO fibroblast cells was accelerated by the immobilization of polyallylamine. The adhesion and growth of the cells were both markedly accelerated by the co-immobilization of the polyallylamine and insulin. The co-immobilized membrane can be applied for a new technique for protein-free cell culture.

Animals↗
PubMedDate not supplied

Prostaglandin E2 restores cancellous bone to immobilized limb and adds bone to overloaded limb in right hindlimb immobilization rats.

The purpose of this study was to determine whether prostaglandin E2 (PGE2) can restore cancellous bone mass and architecture to osteopenic, continuously immobilized (IM), proximal tibial metaphysis (PTM) in female rats. The right hindlimb of three and one-half-month-old Sprague-Dawley female rats were immobilized by right hindlimb immobilization (RHLI) in which the right hindlimb was underloaded and the contralateral left limb was overloaded during ambulation. After 4 or 12 weeks of RHLI, the rats were treated with 3 or 6 mg PGE2/kg/day and RHLI for 8 or 16 weeks. Bone histomorphometry was performed on microradiographs of PTM. Immobilization (IM) induced a transient cancellous bone loss and decreased trabecular thickness, number and node density, and increased free end density that established a new steady state after 4 weeks of IM. Three or 6 mg PGE2/kg/d for 8 weeks beginning at 4 or 12 weeks of IM completely restored cancellous bone mass (+127% to +188%) and structure to the age-related control levels in spite of continuous IM. Another 8 weeks of treatment maintained bone mass and architecture at these levels. No differences in cancellous bone mass and architecture were found between the overloaded PTM or RHLI rats and the age-related controls. However, 3 and 6 mg/kg/d of PGE2 treatment started at 4 or 12 weeks for 8 weeks significantly increased cancellous bone mass in the overloaded PTM (+45 to +74% of untreated controls), and another 8 weeks of treatment maintained bone mass at these levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗
PubMed2001

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization.

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.

Journal Article↗
PubMed1991

Immobilization of yeast and the function of immobilized yeast.

Hansenula anomala, which catalyzes an asymmetric reduction, was immobilized in bulk or spherical crosslinked polymers. The catalytic activity of the yeast for enantio-selective reduction of 3,8-dioxo-4-methoxycarbonyl-9-methyl-delta 4(10)-octalin (lab) was severely affected by the immobilization conditions, such as the crosslink density, and by hydrophobicity of polymers and other components used for immobilization, oxygen concentration in the medium, etc. In some immobilized systems, the ratio of resulting enantiomers was inverted from that in a free yeast system.

Cross-Linking Reagents↗
PubMed1998

Binding specificities of lectins to immobilized glycoproteins and oligosaccharides differ from those of immobilized lectins to oligosaccharides.

The carbohydrate-binding specificities of lectins in solution to glycoproteins and neoglycolipids immobilized on a solid phase were analyzed in order to establish a simple, rapid method for structural analysis of the carbohydrate moieties of small amounts of individual glycoproteins blotted on membrane. Eight glycoproteins containing typical O-linked tetrasaccharides or a series of typical N-linked oligosaccharides of the high-man-nose type, hybrid type, and complex type and 6 neoglycoproteins containing mono- or di-saccharides were dot blotted on membranes and the membranes were then reacted with 8 kinds of horseradish peroxidase-conjugated lectins before and after heat treatment. Neoglycolipids containing the glycoprotein-derived oligosaccharides immobilized on a thin layer chromatography plate were also reacted with lectins. The heat treatment of the membrane increased lectin reactivity toward the glycoproteins. The carbohydrate-binding behavior of lectins, Phaseolus vulgaris erythroagglutinin, wheat germ agglutinin, and concanavalin A in solution toward glycoproteins and neoglycolipids immobilized on a solid phase differed from that of immobilized lectins toward oligosaccharides in solution. This difference should be noted in lectin detection of specific carbohydrates of individual glycoproteins on membrane.

Glycoproteins↗
PubMed2000

Affinity chromatography on immobilized "biomimetic" ligands. Synthesis, immobilization and chromatographic assessment of an immunoglobulin G-binding ligand.

A synthetic bifunctional ligand (22/8) comprising a triazine scaffold substituted with 3-aminophenol (22) and 4-amino-1-naphthol (8) has been designed, synthesised, characterised and immobilized on agarose beads to create a robust, highly selective affinity adsorbent for human immunoglobulin G (IgG). Scatchard analysis of the binding isotherm for IgG on immobilized 22/8 (90 micromol 22/8/g moist weight gel) indicated an affinity constant (Ka) of 1.4 x 10(5) M(-1) and a theoretical maximum capacity of 151.9 mg IgG/g moist weight gel. The adsorbent shows similar selectivity to immobilized protein A and binds IgG from a number of species. An apparent capacity of 51.9 mg human IgG/g moist weight gel was observed under the experimental conditions selected for adsorption. Human IgG was eluted with glycine-HCl buffer with a recovery of 67-69% and a purity of 97.3-99.2%, depending on the pH value of the buffer used for elution. Preparative chromatography of IgG from human plasma showed that under the specified conditions, 94.4% of plasma IgG was adsorbed and 60% subsequently eluted with a purity of 92.5%. The immobilized ligand was able to withstand incubation in 1 M NaOH for 7 days without loss of binding capacity for IgG.

Animals↗
PubMed1992

Anti-IgG immobilized controlled-pore glass. Thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix.

Rabbit anti-bovine IgG was covalently immobilized on thionyl chloride-activated succinamidopropyl controlled-pore glass (CPG) beads (3000 A pore diam; 120/200 mesh). Thionyl chloride-activated beads remained stable for over 1 y retaining full capability of immobilizing protein upon recirculation of protein solution. The immobilized anti-bovine IgG is capable of binding IgG with a dissociation constant (Kd) of 9.45 x 10(-7) M and a capacity of 0.85 g/L of matrix. A column of immobilized anti-IgG is able to remove all detectable contaminating IgG from partially purified enzyme preparations of sulfhydryl oxidase and gamma-glutamyltransferase as determined by ELISA and Western blot. The column matrix could be regenerated by washing with 0.1M acetic acid, pH 2.8.

Animals↗
PubMedDate not supplied

Application of the quantitative sperm immobilization test for follow-up study of sperm-immobilizing antibody in the sera of sterile women.

For quantitative estimation of sperm immobilizing antibody, a new assay method was developed and applied to a follow-up study of antibody titers in the sera of sterile women. The 50% sperm immobilization units (SI50) estimated by the quantitative method ranged from 1.2 to 97.3 in the sera from 16 sterile women who showed relatively high antibody activities in the previous semiquantitative sperm immobilization test. When the quantitative antibody titers, SI50, were followed over 3 years in sterile women with the sperm immobilizing antibody, the antibody titers were found to be unstable, and undulated over a period of several months. Therapy consisting of use of a condom seemed to be ineffective in decreasing the antibody titers.

Adult↗
PubMed1988

[Optimization of methods for immobilizing antibodies against the carcino-embryonic antigen on insoluble matrices. Equilibrium parameters of the interaction of the immobilized antibodies with the antigen].

To obtain highly efficient immunosorbents for solid-phase immunoassay and affinity chromatography, methods for immobilization of antibodies against the carcino-embryonic antigen (CEA) on insoluble matrices were optimized. The immunosorbents obtained were characterized by equilibrium parameters of the reaction between immobilized anti-CEA and CEA calculated from rather a simple kinetic model. This model describes the interaction of the monovalent antigen with two independent types of binding sites. The role of some amino acid residues of anti-CEA in the interaction with CEA was investigated. The effects of immobilization density and the spacer arm length on the functional properties of the immobilized antibodies were studied. The optimal immunosorbent was used to purify 125I-CEA by immunoaffinity chromatography.

Antibodies, Monoclonal↗
PubMed1980

[Steroid transformation with immobilized microorganisms. III. Degradation of the side chain of cholesterol derivatives with immobilized Mycobaterium phlei and M. smegmatis cells].

The cholesterol derivatives 3,3-ethylendioxycholest-5-en I and 4-cholesten-3-(O-carboxymethyl)oxime II were transformed into the corresponding androstenedione derivatives by selective cleavage of the sterol side chain with immobilized preparations of Mycobacterium phlei IMET SG 1026 and M. smegmatis IMET H 124. The influence of three immobilization methods on fermentation activity and stability was investigated. The immobilized cells of M. phlei IMET SG 1026 were found to be suitable to transform the structure modified sterol II in a periodic process over a long period into the androstenedione in a yield of 50--70%. Immobilized cells of M. phlei have higher transformation stability in comparison with free intact cells.

Adsorption↗
PubMed1979

Hydrolysis of urea by gelatin-immobilized urease: separation of kinetic and diffusion phenomena in a model immobilized-enzyme reactor system.

Experiments and appropriate mathematical models are presented in an attempt to elucidate and separate the effects of mass transfer and immobilization on the apparent kinetics of hydrolysis of urea by urease immobilized within a crosslinked gelatin film. Diffusion of urea through the gelatin matrix appears to exert the major influence on the observed kinetics. Diffusion coefficients are measured, and a model for the "effectiveness factor" is presented, accounting for this aspect of mass transfer control. A secondary, but significant, influence on apparent kinetics arises because the reaction products lead to an increased pH level which, because of diffusion resistance, remains high within the gelatin matrix. For pH levels in the 6.7 to 9.0 range the activity of urease is a strongly decreasing function of pH. An approximate model accounting for ionic equilibrium allows this pH-diffusion effect to be introduced in such a way as to lead to predictions of the apparent kinetics that are compared with experimental observations. Examination of these results indicates that the immobilization procedure leads to some loss of activity due to an interaction of the gelatin crosslinking reaction with the enzyme itself.

Cross-Linking Reagents↗
PubMed1977

Immobilization of urea cycle enzymes. I. Characterization of immobilized carbamoylphosphate synthetase and ornithine carbamoyltransferase.

Carbamoylphosphate synthetase (EC 2.7.2.5) and ornithine carbamoyl-transferase (EC 2.1.3.3) extracted from frog liver were successfully immobilized on CNBr-activated Sepharose 4B. The immobilized preparation had a better stability towards heat. The apparent Michaelis constant values for N-acetylglutamate, ammonia, and ATP were not significantly changed by immobilization.

Animals↗
PubMed1992

Enzyme immobilization on a low-cost magnetic support: kinetic studies on immobilized and coimmobilized glucose oxidase and glucoamylase.

Glucose oxidase (GOx) and glucoamylase (GA) were immobilized and coimmobilized through their carbohydrate moieties onto polyethyleneimine-coated magnetite crosslinked with glutaraldehyde and derivatized with adipic dihydrazide. The carbohydrates were oxidized with sodium periodate, and at optimal concentration, their Vm increased up to 18% for GOx and up to 16% for GA. After immobilization, a remaining activity as high as 88% and 70% for GA with maltose and maltodextrin respectively as substrates was obtained, independently of the particle loading. On the contrary, the remaining activity of GOx strongly decreased at high particle loading. Nevertheless, half of its initial activity was recovered at low loading and was not significantly affected when GA was coimmobilized by saturating the reactive groups left on the particle. The Vm of both immobilized enzymes was improved by crosslinking their carbohydrates with adipic dihydrazide, a treatment which allows further coimmobilization of the other enzyme on a second layer.

Enzymes, Immobilized↗
PubMed1980

Application of immobilized enzymes to clinical analyses : use of co-immobilized glucose oxidase and peroxidase in column form.

Glucose oxidase from Aspergillus niger and peroxidase from horseradish were simultaneously immobilized onto alkylamine glass beads which were then packed into a 1.5 x 20 mm column and integrated in the flow system of an AutoAnalyzer I. Glucose in serum, up to 5.0 g/l, was continuously determined at a rate of 60 samples per hour. The co-immobilized enzyme column gave better sensitivity as compared with an enzyme column which contained a mixture of two kinds of individually immobilized enzymes. The enzyme column was sufficiently stable while in use for two months. The results correlated satisfactorily well with those obtained by other well established methods for glucose assay.

Blood Glucose↗
PubMed2002

Advantages of using non-isothermal bioreactors in agricultural waste water treatment by means of immobilized urease. Study on the influence of spacer length and immobilization method.

The behavior of three different catalytic membranes, obtained by immobilizing urease on nylon sheets chemically grafted with methyl methacrylate, was studied in a bioreactor operating under isothermal and non-isothermal conditions. Membrane activation was carried out by condensation or acyl azide reaction, and spacers of different lengths, such as hexamethylendiamine or hydrazine, were used. Under isothermal conditions, the activities of the catalytic membranes and soluble urease were characterized as a function of pH, temperature, and urea concentration. Both enzyme forms showed the same optimum pH, whereas the optimum temperature was lower for the immobilized enzymes. The spacer length appeared to determine broader pH- and temperature-activity profiles for the urease derivatives. The apparent K(m) values of the insoluble urease were dependent on membrane type and were higher than those of the soluble counterpart, thus indicating an affinity loss for urea. Under non-isothermal conditions, all membranes exhibited an increase of percentage activity proportional to the applied temperature difference and decreasing with the increase of urea concentrations. A decrease of the apparent K(m) was also observed. These results suggest that substrate diffusion limitations due to the immobilization process can be overcome in the presence of temperature gradients. In addition, the remarkable reduction of the production times supports the use of non-isothermal bioreactors for the treatment of urea-polluted waste waters.

Agriculture↗
PubMed2005

Impact of surface immobilization and solution ionic strength on the formal potential of immobilized cytochrome C.

Four different self-assembled monolayer (SAM) electrode systems were examined electrochemically in order to better understand surface charge effects on the redox thermodynamics of immobilized horse heart cytochrome c (cyt c). Neutralization of protein surface charge upon adsorption on anionic COOH-terminated SAMs was found to cause substantial changes in the formal potential, as determined by cyclic voltammetry. For cyt c immobilized on negatively charged surfaces, the formal potential shifted to more negative values as the ionic strength was decreased, which is opposite to the trend displayed by solution cyt c. In contrast, immobilization to uncharged interfaces resulted in an ionic strength dependence for cyt c that is similar to its solution behavior. The results provide insight into the importance of surface charge on the formal potential of cyt c.

Adsorption↗
PubMed1993

Immobilization of isochorismate hydroxymutase. Comparison of native versus immobilized enzyme.

Partially purified isochorismate hydroxymutase (isochorismate synthase, E.C. 5.4.99.6) from Flavobacterium K3-15, a vitamin K overproducer, was immobilized on CNBr-activated Sepharose 4B, alkylamine glass substituted with glutardialdehyde, and aminohexyl Sepharose 4B substituted with glutardialdehyde. The immobilized enzyme exhibited a lower specific activity but a broader pH tolerance and a higher thermostability than the soluble enzyme. The stability of the enzyme was greatly increased by immobilization. Isochorismic acid, which is not commercially available, was prepared by a constant flow incubation.

Catalysis↗