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PubMedDate not supplied

Evaluation of beta-galactosidase activity in tissue in the presence of blood.

The reporter gene for beta-galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used beta-galactosidase substrates, including o-nitrophenyl-beta-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of beta-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of beta-galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the beta-galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous beta-galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying beta-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency.

Animals↗
PubMed2004

Optimization of an industrial biocatalyst of glutaryl acylase: stabilization of the enzyme by multipoint covalent attachment onto new amino-epoxy Sepabeads.

Glutaryl-7-aminocephalosporanic acid acylase (GA), an industrially relevant enzyme, has been immobilized onto very different supports, including glyoxyl agarose, heterofunctional epoxy Sepabeads, glutaraldehyde and cyanogen bromide (CNBr) activated supports. Immobilization onto amino-epoxy Sepabeads rendered the most thermo stable preparation of GA, with a half-life time eight times higher than the soluble enzyme, keeping 80% of the enzyme activity. Several parameters that affect the enzyme-support interaction (pH and incubation time) were studied. It was found that after immobilization onto amino-epoxy Sepabeads, incubation at alkaline pH and low temperature exerted dramatic stabilizing effects, increasing the half-life time of the derivative 130 times with respect to the soluble enzyme, while keeping unaltered its intrinsic activity. The loading capacity of the amino-epoxy Sepabeads proved to be very good with a maximum load of 62 mg of protein per g of support with 85 IU/g at 25 degrees C and 200 IU/g at 37 degrees C which makes it a biocatalyst of possible industrial application.

Adsorption↗
PubMed1975

Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli.

Two aminotransferases from Escherichia coli were purified to homogeneity by the criterion of gel electrophoresis. The first (enzyme A) is active on L-aspartic acid, L-tyrosine, L-phenylalanine, and L-tryptophan; the second (enzyme B) is active on the aromatic amiono acids. Enzyme A is identical in substrate specificity with transaminase A and is mainly an aspartate aminotransferase; enzyme B has never been described before and is an aromatic amino acid aminotransferase. The two enzymes are different in the Vmax and Km values with their common substrates and pyridoxal phosphate, in heat stability (enzyme A being heat-stable and enzyme B being heat-labile at 55 degrees) and in pH optima with the amino acid substrates. They are similar in their amino acid composition, each enzyme appears to consist of two subunits, and enzyme B may be converted to enzyme A by controlled proteolysis with subtilsin. The conversion was detected by the generation of new aspartate aminotransferase activity from enzyme B and was further verified by identification by acrylamide gel electrophoresis of the newly formed enzyme A. The two enzymes appear to be products of two genes different in a small, probably terminal, nucleotide sequence.

Amino Acids↗
PubMed2002

Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs.

Pressured by antibiotic use, resistance enzymes have been evolving new activities. Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied. When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins. The X-ray crystallographic structures of three mutant enzymes were determined. These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes. In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr). This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site. This regained stability conferred an advantage in vivo. This pattern of stability loss and restoration may be common in the evolution of new enzyme activity.

Cephalosporins↗
PubMed1996

The C-terminal extension of yeast seryl-tRNA synthetase affects stability of the enzyme and its substrate affinity.

Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes. A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell. However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates. The truncation mutant binds tRNA with 3.6-fold higher affinity, while the Km for serine is 4-fold increased relative to the wild-type SerRS. This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.

Amino Acid Sequence↗
PubMed1995

The effect of low temperatures on enzyme activity.

The stability of two enzymes from extreme thermophiles (glutamate dehydrogenase from Thermococcales strain AN1 and beta-glucosidase from Caldocellum saccharolyticum expressed in Escherichia coli) has been exploited to allow measurement of activity over a 175 degrees C temperature range, from +90 degrees C to -85 degrees C for the glutamate dehydrogenase and from +90 degrees C to -70 degrees C for the beta-glucosidase. The Arrhenius plots of these enzymes, and those for two mesophilic enzymes (glutamate dehydrogenase from bovine liver and beta-galactosidase from Escherichia coli), exhibit no downward deflection corresponding to the glass transition, found by biophysical measurements of several non-enzymic mesophilic proteins at about -65 degrees C and reflecting a sharp decrease in protein flexibility as the overall motion of groups of atoms ceases.

Animals↗
PubMed1983

Alterations in the rates of synthesis and degradation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase produced by cholestyramine and mevinolin.

Hepatocytes were prepared from rats previously fed either a normal diet or diets containing cholestyramine or cholestyramine plus mevinolin. The rates of synthesis and degradation of HMG-CoA reductase were determined by immunoprecipitation of the enzyme from cells radiolabeled with [35S]methionine during standard pulse or pulse-chase experiments. In cells prepared from rats fed either a normal diet or a diet supplemented with cholestyramine or cholestyramine plus mevinolin, the relative rates of reductase synthesis were 0.08, 0.21, and 0.99%, respectively, of the rates of total cellular protein synthesis. The corresponding apparent half-lives of the enzyme were approximately 114 min, 153 min, and greater than 10 h. We propose that the elevated reductase activity observed in rats fed cholestyramine and mevinolin (Tanaka, R. D., Edwards, P. A., Lan, S.-F., Knoppel, E. M., and Fogelman, A. M. (1982) J. Lipid Res. 23, 1026-1031) is a result of both increased enzyme synthesis and stabilization of the enzyme. We demonstrate that enzyme stabilization is not an inherent property of the enzyme but is dependent on the presence of mevinolin. We propose that enzyme stabilization results from the decreased cellular levels of some product produced endogenously from mevalonate.

Animals↗
PubMed2004

Affinity chromatography of porcine pepsin and pepsinogen using immobilized ligands derived from the specific substrate for this enzyme.

Affinity chromatography of porcine protease and its zymogen was carried out on immobilized components of specific substrate used for the pepsin determination. For the immobilization of N-acetyl-L-phenylalanine and iodinated derivative of L-tyrosine, divinyl sulfone activated Sepharose was used. Ligands with blocked amino group and free carboxyl one were linked to Sepharose via ethylene diamine spacer using carbodiimide reaction. Conditions of affinity chromatography of porcine pepsin and pepsinogen on the prepared carriers were optimized: the effect of pH, ionic strength and a nature of the buffers used on adsorption of the enzyme and zymogen to an affinity carrier, as well as their elution was studied. The following parameters were taken into consideration: capacity of the prepared affinity matrices, reproducibility of experiments and the enzyme stability. Pepsin was adsorbed to both immobilized ligands at pH 3.5-4.0; for the elution of the enzyme it was necessary to increase ionic strength (up to 0.5 M). For the adsorption of pepsinogen pH 5.2 was found to be optimum, for its desorption, an increase of ionic strength was used.

Amino Acids↗
PubMed2005

Production and purification of refolded recombinant Plasmodium falciparum beta-ketoacyl-ACP reductase from inclusion bodies.

A recombinant form of Plasmodium falciparum beta-ketoacyl-ACP reductase (PfFabG) was overexpressed in Escherichia coli BL-21 codon plus (DE3). The resulting insoluble inclusion bodies were separated from cellular debris by extensive washing with buffer containing 0.05% Tween 20 and solubilized by homogenization with 8 M urea. Attempts to refold PfFabG from solubilized inclusion bodies employing Rotofor (separation based on different pIs of proteins in a mixture) followed by Ni(2+) or cation exchange chromatography were not successful either by bringing down the urea concentration instantaneously, stepwise, or by dialysis. Denatured PfFabG was therefore initially purified by cation exchange chromatography and was then correctly refolded at a final concentration of 100-200 microg/ml in a 20 mM Na-acetate buffer, pH 5.3, with 300 mM NaCl, 10% glycerol, and 0.05% Tween 20. The protein was found to be properly folded only in the presence of the cofactor NADPH and salt at a concentration 300 mM by drop dilution method at 2-8 degrees C for 12 h. The purified final product was >98% pure by denaturing gel electrophoresis. The purified protein was biologically active in a standard enzymatic assay using acetoacetyl-CoA as a substrate. The enzyme was found to be stable up to fourth day of purification and glycerol was found to stabilize enzyme activity for several weeks, during storage. This effort paves the way for elucidation of the structure-function correlations for PfFabG as well as exploration of the enzyme for developing inhibitors against it for combating malaria.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed1991

Aminoacetone synthase from goat liver. Involvement of arginine residue at the active site and on the stability of the enzyme.

The arginine-specific reagents phenylglyoxal and butane-2,3-dione inactivated goat liver aminoacetone synthase with pseudo-first-order kinetics, with the rate dependent on modifier concentration. Phenylglyoxal and butane-2,3-dione appeared to react with one arginine residue per enzyme molecule. The inactivated enzyme could be re-activated by Tris, suggesting additional evidence of modification of the arginine residue. Acetyl-CoA, one of the substrates, completely protected the enzyme from inactivation. Glycine gave partial protection. Protection by substrates against inactivation by phenylglyoxal and butane-2,3-dione suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of acetyl-CoA showed that only the arginine residue at the active site could be modified by phenylglyoxal. The stability of the enzyme is dependent on the presence of both EDTA and Mg2+.

Acetyl Coenzyme A↗
PubMed1985

Effects of estrogen and tamoxifen on the regulation of dihydrofolate reductase gene expression in a human breast cancer cell line.

We have studied the effects of estrogen and the antiestrogen tamoxifen on the regulation of dihydrofolate reductase (DHFR) gene expression in a methotrexate-resistant (MTXR) human breast cancer cell line MCF-7, which contains a 50-fold increase in the level of DHFR enzyme and amplified DHFR gene sequences. Despite their selection for methotrexate resistance, the MTXR cells have retained many characteristics of the parental MCF-7 cell line. Concentrations of estrogen receptors as well as their binding affinity to estradiol are identical in both cell lines. MTXR MCF-7 cells remain sensitive to estrogen and respond to estradiol with an induction of progesterone receptors, as well as increases in the rate of DNA synthesis and cell growth. Incubation of MTXR MCF-7 cells with estradiol results in an additional 1.5- to 3.0-fold increase in their already elevated level of DHFR. The hormone-induced increases in DNA synthesis and DHFR levels are similar both with respect to the time course of inductions, as well as their dose response to estradiol. However, these two estrogen-induced effects are not coupled, since the induction of DHFR occurs even in the absence of concomitant DNA synthesis. Estradiol has no effect on DHFR enzyme stability; thus, the entire effect of estrogen on DHFR levels results from the increased synthesis of this housekeeping enzyme. In contrast, treatment of MTXR MCF-7 cells with the antiestrogen tamoxifen reduces the rate of DHFR enzyme synthesis, resulting in lower cellular levels of DHFR. These MTXR MCF-7 cells represent a useful model in which to study the mechanisms involved in the modulation of DHFR gene expression by estrogen and tamoxifen. Since the level of DHFR is a critical determinant of methotrexate cytotoxicity understanding, the regulation of DHFR gene expression may have clinical implications for the use of hormonal therapy in combination with chemotherapy for the treatment of breast cancer.

Breast Neoplasms↗
PubMed2000

Increased stability and catalytic efficiency of yeast hexokinase upon interaction with zwitterionic micelles. Kinetics and conformational studies.

The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micelles of lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammonium propanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at 35 degrees C. The thermal inactivation kinetics followed one-exponential decay. The effect of ligands on protecting the enzyme against inactivation followed the order: glucose > glucose/Mg2+ >ATP/Mg2+ approximately or approximately equal to Mg2+l approximately or approximately equal to buffer only. Both LPC and HPS micelles increased the enzyme stability only when the incubation medium contained glucose or glucose/Mg2+, suggesting that the protein conformation is a key prerequisite for the enzyme-micelle interaction to take place. This enzyme-micelle interaction resulted in an increased catalytic efficiency (with a decrease in Km for ATP and increase in Vmax as well as in changes on the tertiary (intrinsic fluorescence) structure of the yeast hexokinase.

Adenosine Triphosphate↗
PubMed1987

Mutations affecting transport and stability of lysosomal enzymes.

The biosynthesis, post-translational processing and receptor-mediated transport of lysosomal enzymes will be briefly summarized. Mutations affecting the transport or the stability of a lysosomal enzyme but not its catalytic properties can result in a lysosomal storage disorder. Mutations causing a loss of catalytic activity may in addition affect transport or stability. Such mutations should not be classified as transport or stability mutations. Prototypes for transport and stability mutations are I-cell disease and late onset forms of metachromatic leukodystrophy.

Carrier Proteins↗
PubMed1989

Induction of rat hepatic N-nitrosodimethylamine demethylase by acetone is due to protein stabilization.

The N-nitrosodimethylamine demethylase (P450I-IE1) is induced severalfold in liver by giving rats ethanol, acetone, pyrazole, and other related small molecular weight compounds. This induction is not the result of an increase in IIE1 mRNA, but could be due to either an increase in translation rate or a decrease in protein degradation. To determine the mechanism of induction, we measured IIE1 synthesis and degradation rates in untreated and acetone-treated rats. This was accomplished by immunopurification of radiolabeled IIE1 protein using a specific monoclonal antibody subsequent to in vivo labeling of total cellular protein with either NaH14CO3 or [3H]leucine. We found that in rats fed acetone, the rate of IIE1 synthesis was not changed; however, IIE1 degradation was markedly altered. In untreated rats, IIE1 protein was degraded via a biphasic pathway consisting of both a rapid and slow component with approximate half-lives of 7 and 37 h, respectively. However, in acetone-treated rats, only a monophasic curve with a half-life of 37 h was observed. The abolition of the rapid degradation component of the IIE1 turnover cycle indicates that induction of IIE1 by acetone is primarily due to specific stabilization of IIE1 protein. Since acetone is also metabolized by IIE1, we believe that this may be a substrate-induced enzyme stabilization.

Acetone↗
PubMed1981

Antimalarials.

The antimalarials, chloroquine, hydroxychloroquine, and quinacrine, are used primarily for malaria; but they can be beneficial for cutaneous lupus erythematosus (LE), polymorphous light eruption, solar urticaria, and porphyria cutanea tarda. Antimalarials bind to deoxyribonucleic acid (DNA) which prevents DNA and ribonucleic acid (RNA) polymerase reactions and DNA heat inactivation; and they inhibit the LE cell phenomenon, antinuclear antibody reactions, and suppress lymphocyte transformation. By competing with calcium ion, they stabilize membranes and have an anesthetic effect. Their anti-inflammatory potential is due to their inhibition of hydrolytic enzymes, stabilization of lysosomes, interference with prostaglandin synthesis, blocking of chemotaxis, and antagonism of histamine responses. The antimalarials have no sunscreening properties. The most common toxic effects are cutaneous pigmentation, nausea, vomiting, diarrhea, mild ileus, and cycloplegia. There has been a reluctance to use chloroquine and hydroxychloroquine because of the possibility of retinopathy. However, if the "safe" daily dose limit of chloroquine, 2 mg per pound of body weight, and of hydroxychloroquine, 3.5 mg per pound of body weight, is followed, the chance of retinopathy is slight. Quinacrine does not cause retinopathy, but it has more cutaneous side effects than the other two agents.

Antimalarials↗
PubMed1992

Enhancement of the stability of thrombin by polyols: microcalorimetric studies.

Glycerol increased the transition temperature (Tm) of thrombin in a concentration-dependent fashion up to a concentration of 50% glycerol in aqueous buffer solution. Glycerol showed a comparable effect on Tm of trypsin. This effect on Tm of thrombin was not seen in the presence of excess sodium chloride (1.2 M) in aqueous buffer solution. The stabilizing effect of glycerol may be due to increased energy demand to unfold the protein molecule, as reflected by an increase in Tm. This stabilizing effect, as measured by Tm, was seen for other polyols, including sucrose, and was also dependent on the concentration of the stabilizing agent. Microcalorimetry may be used as an effective tool to screen for the protective action of compounds in enzyme stabilization studies before conducting the time-consuming and expensive stability studies of proteins in the presence of additives under different storage conditions.

Calorimetry↗
PubMed1989

delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is a multifunctional peptide synthetase.

A multienzyme catalyzing the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the first free intermediate in penicillin biosynthesis, was detected in an assay measuring the formation of tripeptide from L-[U-14C]valine in the presence of L-alpha-aminoadipic acid, L-cysteine, ATP, Mg2+ ions, and dithioerythritol. Enzyme was extracted from dry mycelium using a buffer with a high glycerol concentration and thiol protective agent to stabilize enzyme activity. In five steps the enzyme was purified 118-fold. It catalyzed ATP-pyrophosphate exchange in dependence of all three constituent amino acids, and the enzyme could be amino-acylated with L-[14C]valine. The molecular weight of the protein both native (in gel filtration chromatography) and denatured (polyacrylamide gel electrophoresis) was about 220 kDa. These data suggest that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase consists of a single polypeptide chain and a multienzyme thiotemplate mechanism for the reaction sequence is postulated.

Aspergillus nidulans↗
PubMed1992

Fumarylacetoacetase activity in cultured and non-cultured chorionic villus cells, and assay in two high-risk pregnancies.

We describe further development of the fumarylacetoacetase (FAA) assay for the prenatal diagnosis of tyrosinaemia type 1 using chorionic villus sampling (CVS). We have established a reference range for FAA activity in cultured villus cells and have confirmed previously reported data on the FAA activity in uncultured chorionic villus cells. This should allow confirmation of results using CVS, without the need for further invasive procedures. We report the FAA enzyme stability at -70 degrees C, +4 degrees C, and at room temperature, and we have shown no obvious difference in enzyme activity with gestational age. We have analysed cultured and non-cultured CVS activity of FAA in two pregnancies at risk of tyrosinaemia type 1. In both, the fetus was designated unaffected, and these results were confirmed postnatally.

Cells, Cultured↗