Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.
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PMID:The development and application of a new affinity partitioning system for enzyme isolation and purification. 137 10

Polymer-enzyme hybrid conjugates modified by a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), have been synthesized. We have investigated the molecular architecture of PIPAAm-enzyme conjugates by preparing two types of PIPAAm-trypsin conjugates, wherein PIPAAm chains are attached by either single-end or multipoint chemistry. A semitelechelic co-oligomer (IDc) was attached to trypsin by single-point conjugation (IDc-trypsin). A copolymer (PIDAAc) consisting of acrylic acid and IPAAm randomly linked in polymer chains was attached to trypsin using multipoint conjugation (PIDAAc-trypsin). Both conjugates exhibited reversible temperature-responsive phase separation. The IDc-trypsin conjugate exhibited phase separation at the same temperature as pure IDc, due to the highly mobile free polymer end group which remains sensitive to small temperature changes. The PIDAAc-trypsin conjugate precipitated at higher temperatures than pure PIDAAc, whose movement was restricted by multiple binding points. Enzyme stability in solution was improved after introduction of PIPAAm chains, which prevented autolysis attributed to conjugate steric hindrance. Stability under repeated temperature cycling was also dependent on the architecture of conjugates; the IDc-trypsin conjugate was more stable than the PIDAAc-trypsin. As a consequence, single-end conjugation of polymer to enzyme provides novel bioconjugate with novel functionality attributed to attached polymer while retaining native biological function with high stability.
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PMID:Effect of molecular architecture of poly(N-isopropylacrylamide)-trypsin conjugates on their solution and enzymatic properties. 874 96

Using chain-transfer polymerization, we have synthesized oligomers of poly(N-isopropylacrylamide) [poly(NIPAAm)] with one carboxyl group at the end of each oligomer chain. The lower critical solution temperature (LCST) of the oligomers is very close to that of homo-poly(NIPAAm) lacking the end carboxyl group. The carboxyl groups were activated in methylene chloride using N,N'-dicyclohexyl-carbodiimide (DCC) and N-hydroxysuccinimide (NHS). A conjugate of trypsin with the preactivated oligomer has been prepared. We studied the effect of oligomer to enzyme (O/E) ratio in the feed on the O/E ratio of the conjugate (the average number of oligomer chains conjugated to one trypsin molecule), assuming that only the primary amino groups of lysine residues and the amino terminal of trypsin would react. The O/E ratio of the conjugate was estimated by determination of the remaining primary amine groups on the trypsin molecule. More than 95% of the conjugate can be recovered by thermally induced precipitation.
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PMID:Synthesis and purification of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. 874 99

Reversible soluble-insoluble oligomer-enzyme conjugates have been prepared by conjugating a thermally sensitive oligomer, poly(N-isopropylacrylamide) [poly(NIPAAm)] to trypsin. The conjugates can catalyze enzymatic reactions in solution and then may be separated from the solution by thermal precipitation. One special feature of the conjugates is that every poly(NIPAAm) chain has only one end attachment to the enzyme, so that the loss of enzymatic activity due to steric hindrance should be minimized. Conjugates with various numbers of oligomer chains per trypsin molecule were prepared. Surprisingly, the conjugates increased in enzymatic activity with increasing oligomer conjugation to the native trypsin. The trypsin active sites in the conjugates were accessible to large molecules, such as soybean trypsin inhibitor (MW = 21,500). The enzyme conjugates were more stable than native trypsin, both in solution and in the precipitated phase. On the other hand, the conjugates lost enzymatic activity faster than native trypsin when the temperature was repeatedly cycled through the lower critical solution temperature (LCST) of the poly(NIPAAm). The recovery of the conjugates by thermal precipitation in each cycle was over 95% even after 14 cycles through the LCST.
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PMID:Unusual properties of thermally sensitive oligomer-enzyme conjugates of poly(N-isopropylacrylamide)-trypsin. 946 62

Novel temperature-sensitive polymers containing glucose units in their backbone were synthesized and covalently conjugated to trypsin. A series of copolymers based on N-isopropylacrylamide (NIPAAm) and glucosyoxylethyl methacrylate (GEMA) were prepared by using 4,4'-azobis(4-cyanovaleric acid) as an initiator, which resulted in one terminal carboxylic acid group per polymer chain. The polymers were conjugated to primary amine groups of trypsin with water-soluble carbodiimide as a coupling agent, which led to a star-shaped conformation. The polymer-enzyme conjugation was confirmed and characterized by size exclusion and reversed-phase chromatography. Almost of all amine groups in trypsin available for the conjugation were consumed and, consequently, a very dense layer of copolymers was actually coated around the enzyme surface. The conjugated enzymes exhibited reversible precipitation/resolubilization behaviors over a wide range of temperatures, depending on the content of GEMA in the copolymer. They also demonstrated no detectable self-digestion (autolysis) process, but the unconjugated enzyme showed very severe autolysis that led to a rapid inactivation in aqueous solution. When bovine serum albumin was used as a substrate, the protein substrate was not attacked by the conjugated enzyme, but completely digested by the unconjugated enzyme. This result was presumably caused by a steric repulsion process of the attached polymer chains around the enzyme toward the protein substrate. However, the enzyme retained sufficient activity against a low molecular weight substrate. Interestingly, the conjugated enzymes demonstrated very peculiar enzyme activity-temperature profiles, with two apparent optimal temperatures, indicating that a temperature-controlled collapse and flocculation of the copolymers around the enzyme surface modulated the mass transfer rates of substrate to the active site of the enzyme. The conjugated enzymes also exhibited improved thermal stability with increasing the amount of carbohydrate units in the polymer chain.
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PMID:Conjugation of trypsin by temperature-sensitive polymers containing a carbohydrate moiety: thermal modulation of enzyme activity. 962 35

Thermally reversible poly(N-isopropylacrylamide) (PIPAAm) was covalently grafted onto tissue culture dishes to allow detachment of cultured cells upon temperature change from physiological to room temperature. In addition the grafted polymer matrix was used to entrap biomolecules such as growth factors either to be released by diffusion early in cell cultures, or remain entrapped and be reversibly exposed to cell receptors. Experiments with model proteins trypsin and insulin show that amount loaded and released depends upon the PIPAAm grafting density. Dishes grafted with 2.5 microgram/cm2 PIPAAm released approximately four times more model protein over 4 h than dishes grafted with 1.8 microgram/cm2. This in vitro drug delivery system can be used to deliver factors to the basal side of cells early in cell culture by providing high local concentrations without high bulk concentration. Cultures of human retinal pigmented epithelium showed higher growth rate on insulin loaded dishes than on controls containing a similar bulk solution concentration. These cultures retained the ability to detach singly or as confluent sheets from the loaded surfaces.
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PMID:Growth factor release from thermally reversible tissue culture substrates. 979 28

Bovine aortic endothelial cells were cultured on surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), in the presence of serum. Cells adhered, spread, proliferated, and reached confluency as observed on ungrafted tissue culture polystyrene dishes. A decrease in culture temperature released cells only from the grafted surfaces without enzymatic or ethylenediaminetetraacetic acid treatment. Upon lowering temperature, the culture surfaces changed from hydrophobic to hydrophilic owing to the hydration of grafted PIPAAm and thus weakened the cell attachment to the dishes. Released cells maintained cell-cell junctions composing monolayer cell sheets. Immunoblotting and immunofluorescence microscopy revealed that fibronectin (FN) was deposited and accumulated on the grafted surfaces during the culture. Furthermore, the deposited FN matrix adhering to cell sheets was also recovered from temperature-responsive surfaces by low-temperature treatment, while trypsin treatment destroyed the matrix. The recovery of FN by low-temperature treatment was as high as by physical scraping with a rubber blade. Temperature-responsive surfaces can provide a novel method to use cultured confluent cell sheets for tissue engineering, and also to elucidate structure and function of deposited extracellular matrix during cell culture.
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PMID:Decrease in culture temperature releases monolayer endothelial cell sheets together with deposited fibronectin matrix from temperature-responsive culture surfaces. 1032 8

Copolymers of N-isopropylacrylamide and N-acryloyl amino acid spacers of varying chain length were synthesized. p-Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (Ki) of these polymers for trypsin decreased, i. e., the efficiency of PABA-trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower Ki than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N-acyl-p-aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. Copyright 1999 John Wiley & Sons, Inc.
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PMID:Enhancing ligand-protein binding in affinity thermoprecipitation: elucidation of spacer effects 1039 80

We have developed a new cell culture substrate grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) using an electron beam irradiation method. These surfaces are hydrophobic in culture at 37 degrees C due to the hydration/dehydration changes intrinsic to PIPAAm at 32 degrees C, and they become highly hydrophilic below 32 degrees C. At 37 degrees C grafted and ungrafted surfaces showed no difference with regard to attachment, spreading, growth, confluent cell density, and morphology of bovine aortic endothelial cells. Stress fibers, peripheral bands, and focal contacts were established in similar ways. After the medium temperature was decreased to 20 degrees C, spread cells lost their flattened morphology, acquiring a rounded cell appearance similar to that of cells immediately after plating. After mild agitation cells floated free from the dish surface without trypsin treatment. Neither cell morphological changes nor cell detachment occurred on ungrafted surfaces. An ATP synthesis inhibitor, sodium azide, and a tyrosine kinase inhibitor, genistein, suppressed cell morphological changes and cell detachment while a protein synthesis inhibitor, cycloheximide, slightly enhanced cell detachment. An actin filament stabilizer, phalloidin, and its depolymerizer, cytochalasin D, also inhibited cell detachment. These findings suggest that cell detachment on grafted surfaces is mediated by intracellular signal transduction and reorganization of the cytoskeleton. While trypsinization causes damage to the cell membrane surface and extracellular matrix proteins, this alternative low temperature treatment is exceptionally noninvasive. The temperature-responsive cell culture surface also should prove useful for investigating the molecular machinery involved in cell-surface detachment.
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PMID:Signal transduction and cytoskeletal reorganization are required for cell detachment from cell culture surfaces grafted with a temperature-responsive polymer. 1039 3

We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.
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PMID:Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity. 1076 49


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