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At least 1,207 records · Page 67Linked to original sources
PubMed1985

Stability and storage characteristics of enzymes in cattle blood.

The stability and storage characteristics were studied of 11 bovine enzymes of potential clinical significance, namely, aldolase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, acetylcholinesterase, creatine kinase, gamma glutamyltransferase, glutathione peroxidase (GSH-Px), alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase and superoxide dismutase (SOD). Enzyme activities in fresh serum were compared with those in plasma containing various anticoagulants including lithium heparin, EDTA and oxalate/fluoride. The same preservatives were assessed for their effects on the whole blood activities of GSH-Px and SOD. Stabilities of enzymes in plasma and serum stored at room (+20 degrees C), refrigerator (4 degrees C) or deep freeze (-20 degrees C) temperatures were also compared. In addition, SOD and GSH-Px activities in samples stored, at the same temperatures, as whole blood or aqueous lysates were monitored.

Acetylcholinesterase↗
PubMed1985

Stability and storage characteristics of enzymes in sheep blood.

The stability and storage characteristics were studied of 11 ovine enzymes of potential clinical significance, namely, aldolase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, acetylcholinesterase, creatine kinase, gamma glutamyltransferase, glutathione peroxidase (GSH-Px), alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase and superoxide dismutase (SOD). Enzyme activities in fresh serum were compared with those in plasma containing various anticoagulants including lithium heparin, EDTA and oxalate/fluoride. The same preservatives were assessed for their effects on the whole blood activities of GSH-Px and SOD. Stabilities of enzymes in plasma and serum stored at room (+20 degrees C), refrigerator (4 degrees C) or deep freeze (-20 degrees C) temperatures were also compared. In addition, SOD and GSH-Px activities in samples stored, at the same temperatures, as whole blood or aqueous lysates were monitored. The results are discussed with particular reference to the differences between sheep and cattle.

Acetylcholinesterase↗
PubMed1977

A method for enhancing the stability of thiol-containing enzymes immobilised on nylon.

The limited stability of immobilised enzymes is a major factor restricting their use in clinical chemistry. The immobilisation procedure described here affords a simple method of enhancing the stability of certain enzymes immobilised on nylon. Studies with alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase have indicated that a significant increase in stability can be obtained using this immobilisation procedure as compared with conventional procedures. As anticipated the immobilisation procedure was unsatisfactory for oxidases such as glucose oxidase.

Alcohol Oxidoreductases↗
PubMed1979

Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.

Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.

Animals↗
PubMed2003

Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme.

We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in (2)H(2)O solvent revealed small changes in residual amide II band intensities at approximately 1,550 cm(-1), indicating that (1)H/(2)H exchange in the wild type is clearly perturbed by the mutations. Decreased (1)H/(2)H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45 degrees C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45 degrees C and shows a higher (1)H/(2)H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40-50 degrees C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5 degrees C higher value.

Alanine↗
PubMed1996

Effect of enzyme organization on the stability of Yates-Pardee pathways.

Yates-Pardee-type metabolic pathways in a heterogeneous cell milieu are modeled as a system of coupled non-linear partial differential equations. A numerical solution to this system is described and some properties of such a physiological system are studied. Confinement with and without a membrane is considered and it is shown how confinement results in an increase in the stability of the metabolite concentrations. These results suggest that the enzyme organization may contribute to the stability of the cellular metabolism.

Cell Compartmentation↗
PubMed1989

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme.

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.

Amino Acid Sequence↗
PubMed1986

Three adenovirus type 8 genome types defined by restriction enzyme analysis: prototype stability in geographically separated populations.

Adenovirus type 8 strains were collected over a 19-year period from eye specimens from patients with keratoconjunctivitis. These strains were divided by restriction enzyme analysis with the endonucleases SalI, HindIII, SacI, KpnI, and SmaI into three genotypic subgroups. The prototype strain (Trim) was found throughout the United States from 1966 through 1985 and also in Taiwan and Greece in the early 1980s. Genotype 8C was identified in an arc from Maryland to Missouri to Alabama from 1971 through 1974. Genotype 8D was found only in an epidemic of eye disease among Vietnamese refugees being resettled in northwest Florida in 1975. Genotypes 8C and D were distinct from genotypes 8A and B described from Japan in 1975 through 1981. Hemagglutination tests with a battery of avian and mammalian erythrocytes did not distinguish between the genotypic subgroups. Similarly, the genotypes could not be differentiated by hemagglutination inhibition or serum neutralization tests with reference prototype antisera. The long-term prevalence of the Trim strain suggests that adenovirus type 8 has greater genetic stability than the other adenovirus types studied to date.

Adenoviridae Infections↗
PubMed1997

Structural and kinetic investigations on the 15-21 and 42-45 loops of muscle acylphosphatase: evidence for their involvement in enzyme catalysis and conformational stabilization.

The structural and catalytic importance of the 15-21 and 42-45 loop residues of the acylphosphatase muscular isoenzyme has been investigated by oligonucleotide-directed mutagenesis. Seven mutants involving conserved residues of the two loops have been prepared and characterized for structural, kinetic, and stability features by using different spectroscopic techniques and compared to the wild-type enzyme. The results are discussed in light of the crystal structure of the highly homologous common type acylphosphatase [Thunnissen et al. (1997) Structure 5, 69-79]. A differential role of the two loops has emerged: the 15-21 and the 42-45 loops appear mainly involved in active site formation and enzyme structural stabilization, respectively. These conclusions are supported by a strong impairment of the catalytic efficiency, in terms of enzymatic activity and substrate binding capability, for most of the 15-21 loop mutants. In particular, the Gly15Ala mutant is completely inactive and displays a native-like overall fold, indicating that the correct geometry of the 15-21 loop is an essential requisite for optimal enzymatic catalysis. Instead, the Gly45Ala mutant, though revealing unchanged catalytic properties, shows a considerably reduced conformational stability, as judged by circular dichroism and 1H NMR spectroscopy. This finding confirms previous results relative to Thr42 and Thr46 residues [Taddei et al. (1996) Biochemistry 35, 7077-7083] underlining the structural importance of the 42-45 loop as a linker for the two beta alpha beta units constituting the overall enzyme structure.

Acid Anhydride Hydrolases↗
PubMed2002

Multiple advantages of capillary zone electrophoresis for exploring protein conformational stability.

This review summarizes the work of our laboratory to explore the use of capillary zone electrophoretic (CZE) methods for the investigation of protein conformational stability. Early CZE works on protein denaturation as well as fundamental and theoretical considerations are discussed. Instrumental aspects of the CE-based approach including general and particular CE requirements are documented. Several aspects dealing with estimation of stability of enzymes (cholinesterases and organophosphate-hydrolyzing enzymes) interacting with organophosphates profusely illustrate the multiple advantages of CZE. The discrimination of parameters controlling the "good compromise" stability/plasticity for allowing functional efficiency of these enzymes is exemplified. Thermal stability, susceptibility to high electric field, alteration of stability by bound ligands and the role of associated cations in metalloenzymes have been successfully investigated.

Acetylcholinesterase↗
PubMed1976

Control of enzyme synthesis and stability during sporulation in Saccharomyces cerevisiae.

Studies were undertaken to understand the control of synthesis, stability and modification of UDP galactose epimerase and DNA-dependent RNA polymerase during sporulation of Saccharomyces cerevisiae. When a pre-induced culture of an inducible strain (wild type) is transferred to sporulation medium, the epimerase is inactivated to an undetectable level within 16 hours. Surprisingly, the addition of cycloheximide, a protein synthesis inhibitor, during sporulation stabilizes the epimerase activity. However, in a constitutive strain, the epimerase continues to be synthesized de novo during sporulation. Since the enzyme is synthesized during both vegatative growth and sporulation constitutively, the controls for synthesis of epimerase must be similar under these physiologically different conditions. After chromatography on DEAE Sephadex, there is no change observed in the elution patterns of RNA polymerase forms extracted from acetate growth vegetative cells, sporulating cells or from mature asci ; in all cases RNA polymerase consists of three forms, Ib, II and III. However, single spore suspension obtained from asci by treatment with zymolase contains a new form with chromatographic properties similar to those of form Ia. Our data suggests that form Ia may be a modification product of from Ib.

Carbohydrate Epimerases↗
PubMed2001

Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation.

How can enzymes be protected against denaturation and proteolysis while keeping them in a fully functional state? One solution is to encapsulate the enzymes into liposomes, which enhances their stability against denaturation and proteases. However, the permeability barrier of the lipid membrane drastically reduces the activity of enzyme entrapped in the liposome by reducing the internal concentration of the substrate. To overcome this problem, we permeabilized the wall of the liposome by reconstitution of a porin from Escherichia coli. In this way, we recovered the full functionality of the enzyme while retaining the protection against denaturation and proteolytic enzymes.

Acetylcholinesterase↗
PubMed1984

Sulfhydryl oxidase in human milk: stability of milk enzymes in the gastrointestinal tract.

Sulfhydryl oxidase (SOX) is present in human milk and in milk from all species that have been studied. The pH optimum of human milk SOX is in the neutral range between 7.0 and 7.5. Human milk SOX is stable in an acid environment: 50% of its activity remains after 1 h at pH 2.5. Acid stability is also characteristic of gamma-glutamyltranspeptidase, another membrane-bound enzyme in skim milk. SOX is resistant to pepsin (4000 U/ml), trypsin (50 micrograms/ml), chymotrypsin (200 micrograms/ml), and to trypsin plus chymotrypsin (25 micrograms each/ml). Milk SOX activity has been detected in the stomach and proximal small intestine contents of suckling rats. Human and bovine SOX are relatively heat stable: 75% of the latter remains after treatment at 62.5 degrees C for 30 min and 65% of the former remains after treatment at 60 degrees C for 10 min. Neither remains after 62.5 degrees C for 30 min.

Animals↗