Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enzyme stabilization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed1984

Long-term stability of enzymes in human serum stored in liquid nitrogen.

We analyzed the stability of the enzymes alpha-amylase (EC 3.2.1.1), alkaline phosphatase (EC 3.1.3.1), alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1), creatine kinase (EC 2.7.3.2), glutamate dehydrogenase (EC 1.4.1.3), gamma-glutamyltransferase (EC 2.3.2.2) and lactate dehydrogenase (EC 1.1.1.27) of a human serum pool during storage in liquid nitrogen for a period of 10 months. Except amylase and creatine kinase, all enzymes were stable. Amylase increased in activity, creatine kinase activity decreased. Therefore, human serum stored at -196 degrees C can be used as satisfactory substitute for lyophilized enzyme control serum in internal quality control and stable enzyme material for optimization of methods.

Alanine Transaminase↗
PubMed2001

Effects of nicotinamide coenzymes on the stability of enzyme activities and proteins in niacin-deficient quail tissues against trypsin treatment.

The stability of liver and muscle enzymes and proteins in niacin-deficient quail towards trypsin treatment in the presence and absence of coenzymes, NAD or NADP, was characterized. The protection of liver dehydrogenases by coenzymes was low when they are subjected to trypsin digestion for 60 min. In contrast, in the muscle there was substantial protection against trypsin inactivation of glyceraldehyde-3-phosphate dehydrogenase by NAD and of 6-phosphogluconate dehydrogenase by NADP. Among all enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against trypsin inactivation by NAD. SDS-polyacrylamide gel electrophoresis demonstrated that muscle proteins from the niacin-deficient group were more substantially protected compared to control and pair-fed groups when liver and muscle extracts were spiked with NAD and subjected to trypsin digestion. Overall results suggest that niacin deficiency exerted specific destabilizing effects on the stability of enzymes and proteins in muscle.

Animals↗
PubMed2004

LEAPT: lectin-directed enzyme-activated prodrug therapy.

Targeted drug delivery to selected sites allows reduced toxicity, enhanced efficiency and interchangeable target potential [Langer, R. (2001) Science 293, 58-59 and Molema, G. & Meijer, D. K. F., eds. (2001) Drug Targeting (Wiley-VCH, Weinheim, Germany)]. We describe a bipartite drug-delivery system that exploits (I) endogenous carbohydrate-to-lectin binding to localize glycosylated enzyme conjugates to specific, predetermined cell types followed by (II) administration of a prodrug activated by that predelivered enzyme at the desired site. The carbohydrate structure of an alpha-L-rhamnopyranosidase enzyme was specifically engineered through enzymatic deglycosylation and chemical reglycosylation. Combined in vivo and in vitro techniques (gamma scintigraphy, microautoradiography and confocal microscopy) determined organ and cellular localization and demonstrated successful activation of alpha-L-rhamnopyranoside prodrug. Ligand competition experiments revealed enhanced, specific localization by endocytosis and a strongly carbohydrate-dependent, 60-fold increase in selectivity toward target cell hepatocytes that generated a >30-fold increase (from 0.02 to 0.66 mg) in protein delivered. Furthermore, glycosylation engineering enhanced the serum-uptake rate and enzyme stability. This created enzyme activity (0.2 units in hepatocytes) for prodrug therapy, the target of which was switched simply by sugar-type alteration. The therapeutic effectiveness of lectin-directed enzyme-activated prodrug therapy was shown through the construction of the prodrug of doxorubicin, Rha-DOX, and its application to reduce tumor burden in a hepatocellular carcinoma (HepG2) disease model.

Animals↗
PubMed2002

Trifluoroethanol increases the stability of Delta(5)-3-ketosteroid isomerase. 15N NMR relaxation studies.

In the equilibrium unfolding process of Delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni by urea, it was observed that the enzyme stability increases by 2.5 kcal/mol in the presence of 5% trifluoroethanol (TFE). To elucidate the increased enzyme stability by TFE, the backbone dynamics of Delta(5)-3-ketosteroid isomerase were studied in the presence and absence of 5% TFE by (15)N NMR relaxation measurements, and the motional parameters (S(2), tau(e), and R(ex)) were extracted from the relaxation data using the model-free formalism. The presence of 5% TFE causes little change or a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in the dimer interface region. However, the majority of the residues exhibit reduced order parameters in the presence of 5% TFE, indicating that high frequency (pico- to nanosecond) motions are generally enhanced by TFE. The results suggest that the entropy can be an important factor for the enzyme stability, and the increase in entropy by TFE is partially responsible for the increased stability of Delta(5)-3-ketosteroid isomerase.

Diffusion↗
PubMed1992

[Enzyme immunoassay stability of alpha tumor necrosis factor in plasma and serum].

With an enzyme immunoassay (ELISA) the stability of tumor necrosis factor alpha (TNF-alpha) has been determined in serum and plasma with and without addition of a protease inhibitor (Aprotinin, Trasylol) 24 hours, as well as seven days after blood collection. It has been observed, that after blood collection the concentration of TNF-alpha decreases first and increases again six hours, (+4 degrees C) respectively twelve hours (-20 degrees C) later. Reasons for that might be: The active form of TNF-alpha is a trimer, the presence of TNF-binding proteins, the presence of TNF-antibodies. Furthermore it has been found, that TNF-alpha has the highest stability in plasma at -20 degrees C and -70 degrees C. The addition of Aprotinin had no significant influence on the stability.

Aprotinin↗
PubMed2005

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports.

In this work, we have used supports activated with m-amino-phenylboronic groups to "reversibly" immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.

Bacterial Proteins↗
PubMed1986

Effect of hydroxyurea treatment on lysosomal membrane stability and enzyme latency in L5178Y cells in culture.

The stability of lysosomes prepared from hydroxyurea (HU) treated or untreated L5178Y cells and exposed to an HU-free iso- or hypoosmotic solution was compared. The data revealed that exposure of the cells to 1-10 mM HU for few hours tends to stabilize lysosomes. Partial protection against this effect could be afforded by catalase and superoxide dismutase. Under the same conditions of HU treatment lysosomal enzyme latency remained similar in HU treated and untreated cells.

Acid Phosphatase↗
PubMed2001

Intrinsic versus extrinsic stabilization of enzymes: the interaction of solutes and temperature on A4-lactate dehydrogenase orthologs from warm-adapted and cold-adapted marine fishes.

We examined the effects of temperature and stabilizing solutes on A4-lactate dehydrogenase (A4-LDH) from warm- and cold-adapted fishes, to determine how extrinsic stabilizers affect orthologs with different intrinsic stabilities. Conformational changes during substrate binding are rate-limiting for A4-LDH, thus stabilization due to intrinsic or extrinsic factors leads to decreased activity. A4-LDH from a warm-temperate goby (Gillichthys mirabilis), which has lower values for kcat and the Michaelis constant for pyruvate ( K m PYR), was intrinsically more stable than the orthologs of the cold-adapted Antarctic notothenioids Parachaenichthys charcoti and Chionodraco rastrospinosus, as shown by a higher apparent transition ('melting') temperature (Tm(APP)). We used four solutes, glycerol, sucrose, trimethylamine-N-oxide and poly(ethylene glycol) 8000, which stabilize proteins through different modes of preferential exclusion, to study temperature-solute interactions of the three orthologs. Changes in Tm(APP) were similar for all orthologs in each solute tested, but the catalytic rate of G. mirabilis A4-LDH was decreased most by solutes and increased most by temperature. In contrast, the K m PYR values of the Antarctic orthologs were more affected than that of the goby by both solutes and temperature. We conclude that (a) preferential exclusion of solutes functions within the native state of A4-LDH to favor conformational microstates with minimal surface area; (b) the varied effects of the different solutes on the kinetic properties are due to the interaction between this nonspecific stabilization and the differing intrinsic stabilities of the orthologs; (c) the catalytic rates of A4-LDH orthologs are equally affected by stabilizing solutes, if measurements are made at physiologically appropriate temperatures; and (d) global stability and localized flexibility of these A4-LDH orthologs may evolve independently.

Animals↗
PubMed1985

Stability of enzymes.

Enzymes can lose activity through covalent and noncovalent structure alterations. In the former, protease attack and modification by small active molecules such as oxygen are important. Conformational stability can be measured by Tm, the midpoint temperature of the thermal denaturation curve, and turnover in vivo of a number of enzymes correlates with Tm. Measurement of Tm and delta Cp leads to evaluation of delta H, T delta S, and delta G for the unfolding process. The importance of d(delta G)/dT is emphasized since it can be used to evaluate the temperature of maximum stability. There is no simple relationship between amino acid sequence and delta Gmax, nor can the effect of mutation be accurately forecast. Reversibility of folding is an important factor in stability. Tm correlates with [G]1/2, the midpoint guanidine unfolding concentration, and is the most useful predictive quantity for enzyme stability.

Animals↗
PubMed1999

The Saccharomyces cerevisiae succinate dehydrogenase anchor subunit, Sdh4p: mutations at the C-terminal lys-132 perturb the hydrophobic domain.

The yeast succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits, Sdh1p-Sdh4p, that couples the oxidation of succinate to the transfer of electrons to ubiquinone. One of the membrane anchor subunits, Sdh4p, has an unusual 30 amino acid extension at the C-terminus that is not present in SDH anchor subunits of other organisms. We identify Lys-132 in the Sdh4p C-terminal region as necessary for enzyme stability, ubiquinone reduction, and cytochrome b562 assembly in SDH. Five Lys-132 substituted SDH4 genes were constructed by site-directed mutagenesis and introduced into an SDH4 knockout strain. The mutants, K132E, K132G, K132Q, K132R, and K132V were characterized in vivo for respiratory growth and in vitro for ubiquinone reduction, enzyme stability, and cytochrome b562 assembly. Only the K132R substitution, which conserves the positive charge of Lys-132, produces a wild-type enzyme. The remaining four mutants do not affect the ability of SDH to oxidize succinate in the presence of the artificial electron acceptor, phenazine methosulfate, but impair quinone reductase activity, enzyme stability, and heme insertion. Our results suggest that the presence of a positive charge on residue 132 in the C-terminus of Sdh4p is critical for establishing a stable conformation in the SDH hydrophobic domain that is compatible with ubiquinone reduction and cytochrome b562 assembly. In addition, our data suggest that heme does not play an essential role in quinone reduction.

Cytochrome b Group↗
PubMed1977

Long-term stability of enzymes, total protein, and inorganic analytes in lyophilized quality control serum.

The stability of total protein and various enzyme and electrolyte analytes in numerous pools of lyophilized quality control sera is evaluated. Using data obtained between 1973 and 1976 from Regional Quality Control Programs utilizing the Quality Assurance Service of the College of American Pathologists, monthly group mean values are studied as a function of time, by means of linear regression analysis. In the 28 pools studied, unstable analyte-pool combinations were detected in approximately 35% of the cases. Total protein was the most universally stable. In a large percentage of pools, minimally increasing concentrations of sodium, chloride, and potassium were found, while inorganic phosphorus decreased in the majority of pools. In pools with changing enzymatic activiteis, alkaline phosphatase tended to increase and creatine phosphokinase generally decreased. Changes in levels of calcium, serum glutamic oxaloacetic transaminase, and lactate dehydrogenase were inconsistent. Changes in analyte concentration were judged in relation to the precision (SD) with which the analytes were measured. The ratios of rate of change in analyte value to SD were lower for electrolytes than for other analytes.

Alkaline Phosphatase↗
PubMed2006

Challenges in biocatalysis for enzyme-based biofuel cells.

Enzyme-based biofuel cells are attracting attention rapidly partially due to the promising advances reported recently. However, there are issues to be addressed before biofuel cells become competitive in practical applications. Two critical issues are short lifetime and poor power density, both of which are related to enzyme stability, electron transfer rate, and enzyme loading. Recent progress in nanobiocatalysis opens the possibility to improve in these aspects. Many nano-structured materials, such as mesoporous media, nanoparticles, nanofibers, and nanotubes, have been demonstrated as efficient hosts of enzyme immobilization. It is evident that, when nanostructure of conductive materials are used, the large surface area of these nanomaterials can increase the enzyme loading and facilitate reaction kinetics, and thus improving the power density of biofuel cells. In addition, research efforts have also been made to improve the activity and stability of immobilized enzymes by using nanostructures. It appears to be reasonable to us to expect that progress in nanostuctured biocatalysts will play a critical role in overcoming the major obstacles in the development of powerful biofuel cells.

Bioelectric Energy Sources↗