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At least 19 recordsLinked to original sources
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↗
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↗
PubMed1979

Immobilization of aminoacylase by adsorption to tannin immobilized on aminohexyl cellulose.

The immobilization of aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated by using tannin immobilized on aminohexyl cellulose. The most active immobilized aminoacylase was obtained when aminoacylase was adsorbed to the immobilized tannin in a weak alkaline medium containing sodium chloride and n-butanol at 37 degrees C. The activity of the immobilized tannin-aminoacylase complex per unit volume was five times higher than that of the DEAE-Sephadex-aminoacylase complex used for industrial production of L-amino acids in our plants. The half-life of the immobilized tannin-aminoacylase complex was 20 days under continuous operation at a high concentration of substrate; on the contrary, that of the DEAE-Sephadex-aminoacylase complex was 0.5 days.

Adsorption↗
PubMedDate not supplied

[Immobilization of Penicillium vitale glucose-oxidase on aminosilochrome and properties of immobilized enzyme].

Penicillium vitale glucose-oxidase modified by means of the carbohydrate component oxidation is added covalently to aminoorganosylochrome. The activity of the immobilized preparations is 20-38% depending on the protein-carrier ration in immobilization. Comparison of some properties of native and immobilized glucose-oxidase showed that the rH optimum of the immobilized glucose-oxidase is slightly widened towards the alkaline regions; the immobilized glucose-oxidase possesses a considerably higher pH-stability at pH alkaline values; the immobilized glucose-oxidase preparations are characterized by a significantly greater thermostability: their thermoinactivation constant at 65 degrees C is 8-10 times lower than that of the native enzyme.

Drug Stability↗
PubMed1977

Immobilization of urea cycle enzymes. II. Characterization of immobilized argininosuccinate synthetase.

Argininosuccinate synthetase (EC 6.3.4.5) was immobilized on CNBr-activated Sepharose 4B. Properties of the immobilized enzyme are described and compared with those of the native enzyme. The immobilized enzyme was much more stable than the native enzyme at 37 degrees C. It was further stabilized in the presence of the assay reagents. The optimum pH of the immobilized enzyme shifted towards alkalinity (approximately 0.5 unit). The apparent Michaelis constants measured for the immobilized enzyme were not greatly different from those measured for the native enzyme. Urea formation from citrulline was confirmed in a continuous column reactor by the coimmobilized argininosuccinate synthetase, argininosuccinate lyase (EC 4.3.2.1), and arginase (EC 3.5.3.1).

Animals↗
PubMed1992

Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: the immobilization of trypsin and alpha-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support.

Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.

Benzoylarginine Nitroanilide↗
PubMed1979

Use of immobilized enzymes in automated clinical analysis: determination of uric acid and glucose using immobilized enzymes in column form.

We studied the use of immobilized enzymes, covalently bound to alkylaminosilane derivative of porous glass, to automated clinical analysis on uric acid and glucose in blood, serum and urine. A microcolumn with an immobilized enzyme was prepared and used in an AutoAnalyzer I continuous flow system. Uricase (EC 1.7.3.3) from Candida utilis and glucose oxidase (EC 1.1.3.4) from Aspergillus niger were immobilized for the determination of uric acid and glucose, respectively. Hydrogen peroxide produced by these oxidases was colorimetrically determined using horse-radish peroxidase (EC 1.11.1.7) and a hydrogen acceptor in solution. Sensitivity and wash charactertistics of a column with immobilized enzyme, 1.5 mm of inner diameter and up to 40 mm in length, were satisfactory at an assay speed of 50 samples per hour. The results correlated well with those obtained by other well established methods utilizing the AutoAnalyzer system. The immobilized enzymes were sufficiently stable for at least two months of 2000 tests when used repeatedly. Clinical trials proved that this method is capable of replacing the soluble enzyme method, giving reliable and reproducible results at lower cost.

Autoanalysis↗
PubMed1976

Studies on the Treponema pallidum immobilizing activity in normal human serum. 3. The kinetics of immobilization reaction of normal and immune sera.

The influence of immobilizing antibody, complement and lysozyme, on the T. pallidum immobilization reactions of normal and immune sera was studied. Lysozyme shortened the lag periods and increased the reaction rates of the reactions of normal and immune sera. At high concentrations of added lysozyme, variations in the concentrations of immobilizing antibody and complement, within a wide range, did not further influence the kinetics of the two reactions. Preincubation with lysozyme did not influence the treponemes in the following immune serum immobiliation reaction provided the lysozyme was removed before the addition of antibody and complement. Normal serum was found to immobilize T. pallidum more rapidly than immune serum. This was seen also if the reaction mixtures were almost identical, the only differences being the immobilizing IgM antibody involved in the normal and the IgG antibody involved in the immune serum reaction.

Blood↗
PubMed1992

Cell growth on immobilized cell-growth factor. II. Adhesion and growth of fibroblast cells on poly(methyl methacrylate) membrane immobilized with proteins of various kinds.

The surface of poly(methyl methacrylate) membrane was partially hydrolysed and the carboxyl groups produced were coupled with various protein molecules with water-soluble carbodiimide. The immobilized proteins were a cell-growth factor insulin, cell adhesion factors fibrinogen and fibronectin, and serum proteins albumin and gamma-globulin. The insulin-immobilized poly(methyl methacrylate) membrane strongly accelerated the growth and slightly accelerated the adhesion of fibroblast cells. The immobilized fibronectin and fibrinogen enhanced the cell adhesion, and the former also accelerated the cell growth. The immobilized albumin and gamma-globulin influenced the adhesion and growth of cells very little. It was found that various proteins specifically influence the adhesion and growth of cells in an immobilized state.

Animals↗
PubMed1976

Studies on the Treponema pallidum immobilizing activity in normal human serum. 2. Serum factors participating in the normal immobilization reaction.

The T. pallidum immobilization reaction to be achieved by unheated normal serum was found to be complement dependent and the results presented suggested that complement was activated via the classical pathway. Besides complement, an immobilizing antibody of the IgM class was necessary for the immobilization reaction to occur. Lysozyme exerted an enhancing effect on the normal serum immobilization reaction.

Blood↗
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↗
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↗
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↗
PubMed1977

Dissociation and reassociation of immobilized porphobilinogen synthase: use of immobilized subunits for enzyme isolation.

The dissociation and association of an immobilized preparation of the octameric enzyme porphobilinogen synthase [5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24] is described. On treatment of the immobilized preparation with 4 M urea, four subunits per octamer are removed which can be reassociated into a soluble octameric enzyme. The tetrameric bound residual protein can also be reassembled into an octameric structure, with the same initial enzyme activity, by exposing the residual bound protein to a soluble pure enzyme preparation or to a crude liver extract in the presence of urea. The dissociation of the reconstituted bound enzyme releases subunits that again can be reassembled into a soluble octameric pure protein even when the crude liver preparation is used as the donor of the subunits. Thus, a pure enzyme can be isolated in a reassociation-dissociation cycle. The use of immobilized preparations of oligomeric proteins is considered for intra- and interspecies hybridization studies and for the ready preparation of purified enzyme preparations from different species and is suggested as a model for study of the formation of an oligomeric enzyme in the presence of other polypeptides.

Animals↗
PubMed1979

Immobilized enzyme electrodes for the potentiometric measurement of glucose concentration: immobilization techniques and materials.

Glucose oxidase, catalase, and bovine serum albumin were co-immobilized with glutaraldehyde around a platinum screen or around a single platinum-iridium wire. The potential difference between this dual enzyme electrode and a Ag/AgCL reference electrode was proportional to the logarithm of the glucose concentration over the range from 10 to about 150 mg glucose per 100 ml in buffered solution at pH 7.4 and 37 degrees C. The enzyme electrode responded in serum only if coated with a semipermeable film, such as cellulose acetate, to exclude serum macromolecules. The potentiometric results were similar to those obtained with the two enzymes co-immobilized in polyacrylamide gel around a platinum screen or with only one of the enzymes, glucose oxidase, covalently coupled to a platinum screen. The results so far suggest that glucose for development of a continuous in vivo glucose sensor.

Catalase↗
PubMed1990

Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel beads: I. Effect of immobilization on the primary and ultimate biodegradation of SDS, and redistribution of bacteria within beads during use.

The surfactant-degrading bacterium Pseudomonas C12B was immobilized in polyacrylamide gel beads. Conditions were established for minimizing the apparent loss of sodium dodecyl sulfate (SDS)-degrading activity accompanying polymerization, while still retaining durable gel beads. Apparent losses in SDS-degrading activity compared with free untreated bacteria were attributed largely to substrate diffusion limitations imposed by the gel matrix. Changes in the rate and extent of conversion of radiolabel from [1-14C]SDS to 14CO2 were attributed to diffusional restrictions on O2 availability within the gel beads. Scanning electron microscopy was used to show that beads (3 mm3) repeatedly exposed to SDS for 35 days contained a high cell density in a sub-surface layer 0.4-0.7 mm deep, with relatively few bacteria either at greater depth or at the bead surface.

Biodegradation, Environmental↗