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At least 847 records · Page 47Linked to original sources
PubMed2006

A new, sensitive method for enzyme kinetic studies of scarce glucosides.

The maize beta-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates. We developed a glucose assay using a system comprising three enzymes beta-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine-Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-beta-d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.

Calibration↗
PubMed1989

Generation of triplet carbonyl compounds during peroxidase catalysed reactions.

Peroxidases, acting as oxidase upon appropriate substrates, generate carbonyl compounds in the electronically excited triplet state. These excited species can transfer energy as demonstrated by the appearance of the acceptor fluorescence or induced photochemistry concomitant with the disappearance of phosphorescence. Chlorophyll, an efficient emissive acceptor, either naturally present or artificially incorporated into organelles and cells, allows the in situ detection of biologically generated excited species. With neutrophils, the myeloperoxidase promoted acetone phosphorescence can readily be detected. In other cases, e.g. triplet benzaldehyde, it is possible to observe emission from lipid peroxidation initiated by the triplet carbonyl compound.

Acetaldehyde↗
PubMedDate not supplied

Inhibition of the myeloperoxidase-H2O2-Cl- system of neutrophils by indomethacin and other non-steroidal anti-inflammatory drugs.

The results presented herein demonstrate that the non-steroidal anti-inflammatory drug (NSAID) indomethacin is a strong inhibitor of the formation of HOCl by murine neutrophils (50% inhibition at 15 microM). Addition of 40 microM indomethacin to activated neutrophils caused 80% inhibition of HOCl formation throughout a 60-min time course while slightly increasing the levels of O2- and H2O2 produced. Comparable degrees of inhibition were achieved when the cells were stimulated with phorbol myristate acetate and with opsonized zymosan. Control experiments indicated that the drug did not act by scavenging HOCl. Direct inhibition of the chlorinating activity of myeloperoxidase (MPO) was confirmed using highly purified human enzyme in vitro. Kinetic analysis of the mechanism of inhibition showed that the drug was competitive with respect to Cl- and uncompetitive with respect to H2O2, showing a Ki of 37 microM. In contrast to its inhibition of the oxidation of Cl- by MPO, indomethacin had no effect on the peroxidative activity of the enzyme (oxidation of 4-aminoantipyrene), nor did it inhibit the activity of several other enzymes involved in H2O2 metabolism, including horseradish peroxidase, catalase, xanthine oxidase, and superoxide dismutase. Finally, it was found that inhibition of HOCl formation was a shared but non-uniform property of many NSAIDs; piroxicam, salicylate, sulindac, ibuprofen, and aspirin were all inhibitory but at widely different concentrations [Ki(app) values of 0.05, 0.18, 0.18, greater than 1, and 3 mM respectively] that correlated only partially with their therapeutic dose range. The results encourage further studies into the possibility that inhibition of HOCl formation may constitute an additional mechanism whereby NSAIDs reduce tissue destruction in chronically inflamed tissues.

Animals↗
PubMedDate not supplied

Caloric and carbohydrate restriction in the kidney: effects on free radical metabolism.

Carbohydrate restriction and caloric restriction (60% restriction of calories in relation to controls in both cases) were imposed on OF1 mice during 8 weeks in their growing phase. The three groups of animals ingested the same amount of vitamins and minerals. Kidney ascorbate strongly decreased in both restriction groups. Nevertheless, global caloric restriction significantly increased kidney antioxidant glutathione (GSH)/oxidized glutathione (GSSG) ratio, a sign of a reduced kidney oxidative stress. Increased glutathione peroxidase and cytochrome oxidase activities and decreased in vivo peroxidation were found in the kidney when the restriction was performed by substituting carbohydrates by nonnutritive bulk. No significant changes were observed for superoxide dismutase, catalase, glutathione reductase, glutathione, uric acid, malondialdehyde (HPLC), or in vitro sensitivity to peroxidation in the kidney. The results, reported for the first time in this tissue, show that short-term caloric restriction can increase the capacity for enzymatic decomposition of hydroperoxides and can decrease oxidative stress in the kidney, thus suggesting a role for free radical metabolism in the caloric restriction phenomenon.

Animals↗
PubMed1996

Amperometric microsensors for monitoring choline in the extracellular fluid of brain.

Selective amperometric enzyme microsensors for monitoring low micromolar concentrations of choline in extracellular fluid of rat brain have been developed. Preparation of the choline microsensors involved the modification of carbon fiber microcylinder electrodes (10 microns diameter, 300-400 microns long) with a cross-linked redox-active gel containing horseradish peroxidase and choline oxidase. Rejection of the noise recorded from the choline microsensors implanted in living brain tissue improved the in vivo detection capabilities of the sensors. The microsensors and a differential detection scheme were used to estimate the basal concentration of choline in striatal tissue at 6.6 +/- 2.9 microM and to measure changes in choline concentrations of 6.1 +/- 2.7 microM in vivo. The microsensors were also used to monitor choline produced following the injections of acetylcholine in vivo. Coinjections of neostigmine and acetylcholine significantly lowered the choline response recorded with the microsensors, confirming that the response following the injections of acetylcholine alone was due to the activity of endogenous acetylcholinesterase. Comparison of the maximal rate of decrease in choline concentration following the injections of 1 mM choline and 1 mM acetylcholine was used to estimate the rate of acetylcholine clearance from extracellular fluid through cholinesterase activity at approx. 2.5 microM/min.

Acetylcholine↗
PubMed2006

Evaluation of antioxidant activity and potential toxicity of 1-buthyltelurenyl-2-methylthioheptene.

The aim of the present study was to evaluate pharmacological and toxicological properties of 1-buthyltelurenyl-2-methylthioheptene (compound 1). In vitro, compound 1 at 1 microM was effective in reducing lipid peroxidation induced by Fe/EDTA. Compound 1 presented neither thiol peroxidase nor thiol oxidase activity and did not change delta-ALA-D (delta-aminolevulinate dehydratase) activity (10-400 microM). Calculated LD(50) of compound 1, administered by oral route, was 65.1 micromol/kg. Rats treated with compound 1 did not reveal any motor impairment in the open field. Hepatic, renal and cerebral lipid peroxidation in treated rats did not differ from those in control rats. Conversely, 0.5 micromol/kg of compound 1 decreased lipid peroxidation in spleen. Delta-ALA-D activity in liver and spleen was inhibited in rats treated with the higher dose of compound 1 but no significant differences were detected in renal delta-ALA-D activity. AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities as well as urea and creatinine levels were increased by high doses of compound 1 (50-75 micromol/kg). Compound 1 induced a significant decrease in plasma triglyceride levels but none of the doses tested changed the cholesterol level. This is a promising compound for more detailed pharmacological studies involving organotellurium compounds.

Alanine Transaminase↗
PubMed1978

The 4-hydroxybenzoate/4-aminophenazone chromogenic system used in the enzymic determination of serum cholesterol.

A single reagent, containing cholesterol oxidase, cholesterol esterase, peroxidase, 4-hydroxybenzoate, and 4-aminophenazone, is used in determining serum cholesterol. Analysis time is 15 min, and the standard curve is linear to 6.0 g/liter. Analytical recovery of cholesterol was 100.1 +/- 0.4%. Within-run precision (CV) was less than or equal to 1.4 1.4%, between-run less than or equal to 4.8%. Comparison with results by a Liebermann Burchard method [Clin. Chim. Acta 5, 637 (1960)] gave a linear regression of y = 1.08x--0.05, with a correlation coefficient (r) of 0.985. Comparison with the Roeschlau enzymic method [J. Clin. Chem. Clin. Biochem, 12, 226 (1974)] gave y = 1.02x + 0.01 (r = 0.958). Comparison with the enzymic method of Allain et al. [Clin. Chem. 20, 470 (1974)] gave y = 1.01x--0.00 (r = 0.995). The following substances do not interfere up to the indicated concentrations (mg/liter): hemoglobin (5000), bilirubin (100), reduced glutathione (150), l-cysteine (400), urea (3000), creatinine (200), uric acid (200), d-glucose (10000), L-ascorbic acid (50), acetylsalicylic acid (500), L-DOPA (10), ergothioneine (1000), 2,5-dihydroxybenzoic acid (20), and 3,4-dihydroxybenzoic acid (10). Stored in an amber-colored bottle, the working reagent is stable for three months at 2--8 degrees C and for three weeks at 25 degrees C.

Aminopyrine↗
PubMed1994

Quantitation of choline in the extracellular fluid of brain tissue with amperometric microsensors.

Amperometric microsensors for the detection of choline in the extracellular fluid of brain tissue have been prepared by immobilizing horseradish peroxidase and choline oxidase onto carbon fiber microcylinder electrodes with a cross-linkable redox polymer. The microcylinders have diameters of 7 or 10 microns and lengths of 200-400 microns. To detect choline, the microsensors are operated at an applied potential of -0.1 V vs SCE. At this potential, ascorbate and other easily oxidizable interferant molecules present in brain tissue are not detected by the electrode. Ascorbate, however, can interfere with the response to choline by acting as a reducing agent in the enzyme-containing polymer film. So, a Nafion overlayer is required in order to reliably detect choline in the presence of physiologically relevant concentrations of ascorbate (approximately 200 microM). The Nafion-coated microsensors have a detection limit of approximately 5 microM choline and give a linear response beyond 100 microM when calibrated in vitro at 37 degrees C. Exposure of the microsensors to brain tissue for several hours causes less than a 10% loss in redox polymer surface coverage and less than a 25% loss in sensitivity to choline. To assess the ability of the microsensors to monitor choline levels in brain tissue, small volumes of a choline solution were injected into brain tissue at a site about 1 mm away from a microsensor. The current arising at the microsensor was converted to choline concentration by calibrating the sensor following the in vivo experiment. The resultant choline concentrations were in excellent agreement with those predicted by appropriate diffusion equations.

Animals↗
PubMed2002

Sol-gel encapsulated enzyme arrays for high-throughput screening of biocatalytic activity.

We developed versatile low-cost arrays of sol-gel-encapsulated enzymes (referred to as solzymes) suitable for repeated assays of bioactivity or enzyme inhibition. Sol-gel microstructures containing active enzymes were stabilized on glass at moderate pH and room temperature without harsh calcination. A multi-well bilayer of polydimethylsiloxane was used to support the solzyme array and contain the reaction medium. Each of the 147 microwells has a working volume of 5 muL and contains 50 mug of immobilized enzyme. The solzyme arrays maintained high activity through repeated applications and exhibited superior thermostability compared to soluble enzymes. Among the enzymes used were lipases, glucose oxidase, and horseradish peroxidase. Twenty different lipases and proteases were also used to prepare a hydrolase array, for which bromthymol blue served as a generic indicator of activity. The relative activities of the encapsulated hydrolases correlated closely with those of the soluble hydrolases, illustrating that sol-gel encapsulation preserved the hierarchy of enzyme activity. The development of solzyme arrays paves the way to higher throughput screening of diverse proteins and enzymes, including those that are available only in trace amounts.

Biosensing Techniques↗
PubMed2003

Relative messenger RNA abundance in bovine oocytes collected in vitro or in vivo before and 20 hr after the preovulatory luteinizing hormone surge.

In the cyclic cow, final maturation of the ovulatory follicle is initiated by the preovulatory luteinizing hormone (LH) surge. During the subsequent 24 hr period, the oocyte nucleus undergoes meiotic progression to metaphase II and several changes in cytoplasmic organization take place. We have previously shown that oocytes recovered at the time of the LH peak and matured in vitro are less competent to reach the blastocyst stage than their counterparts recovered 20 hr later following in vivo maturation, despite both groups undergoing IVF and culture in parallel. The objective of this study was to compare, using real-time quantitative RT-PCR, the relative abundance of various developmentally important gene transcripts in these oocytes. The groups used were mature bovine oocytes originating from: (1) 2-6 mm follicles from slaughterhouse ovaries; (2) preovulatory follicles punctured by ovum pick-up just before the LH surge (i.e., immature) and matured in vitro; or (3) preovulatory follicles punctured 20 hr later, just prior to ovulation (i.e., in vivo matured). In addition, immature oocytes from 2-6 mm follicles were examined. We examined the relative mRNA expression of five enzymes involved in protection against free oxygen radicals (mitochondrial Mn-superoxide dismutase, MnSOD, cytosolic Cu/Zn superoxide dismutase, Cu/ZnSOD, gamma-glutamyl-cysteine transferase, GCS, glutathione peroxidase, GPX, sarcosine oxidase, SOX), a transcript involved in follicular development (growth differentiation factor-9, GDF-9), transcripts involved in glucose metabolism (glucose-6-phosphate dehydrogenase, G6PDH, glucose transporter type-1 and -8, Glut-1, Glut-8) and genes involved in cell cycle events, Cyclin A and B, and poly(A) polymerase (PAP). Transcripts for all genes were detected, irrespective of oocyte origin. While differences were not significant in all cases, variations in levels of transcript abundance between the groups were related to developmental competence. In particular, transcripts for GDF-9 were expressed at significantly higher levels in oocytes recovered at the LH peak and matured in vitro than in those matured in vivo. The observations with GDF-9 are interesting as this gene is believed to be essential for normal folliculogenesis and may be important in the regulation of early follicle and oocyte growth. In conclusion, the results of this study demonstrate differences in the relative mRNA abundance of several developmentally important gene transcripts in bovine oocytes which may be related to developmental competence.

Animals↗
PubMed1996

Enzyme-coupled assays for proteases.

We have developed a general strategy for assaying proteases that does not require the use of fluorogenic, chromogenic, or radiolabeled peptide substrates. The endo- or exoproteolytic hydrolysis of simple peptides can be followed spectrophotometrically by coupling the proteolytic event via enzyme-catalyzed reactions to a chromogenic redox dye. The couple can be used directly to follow the action of carboxy or amino peptidases on peptide substrates or can be coupled by use of carboxy or amino peptidases to follow the action of endoproteases on peptide substrates that are blocked at the amino or carboxy terminus, respectively. Liberated amino acids are detected by use of amino acid oxidase, oxygen, horseradish peroxidase, and the redox dye 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid (epsilon 414nm = 36,000 M-1 cm-1).

Biological Assay↗
PubMed2000

N-Glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1.

A method has been developed which allows the analysis of glycoproteins separated by SDS-PAGE. The procedure, though applicable to N-glycosylated glycoproteins of any origin, is particularly devised for glycoproteins potentially containing fucose in alpha1,3-linkage to the reducing GlcNAc as may be found in plants and invertebrates, e.g., insects and parasitic helminths. Starting with an established procedure for mass spectrometric peptide mapping, the analysis of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry involved the use of peptide:N-glycosidase A, a triphasic microcolumn for sample cleanup, and a new matrix mixture consisting of 2,5-dihyhydroxybenzoic acid, 1-hydroxyisoquinoline, and arabinosazone. The method was tested on proteins with N-glycans of known structure, i.e., as horseradish peroxidase, zucchini ascorbate oxidase, soybean agglutinin, honeybee venom hyaluronidase, bovine ribonuclease B, and bovine fetuin. An electrophoretic band corresponding to 4 microg of glycoprotein was generally sufficient to allow detection of the major N-glycan species. As an additional benefit, a peptide mass map is generated which serves to identify the analyzed protein. The method was applied to glycoprotein allergens whose glycan structures were unknown. Ara h 1 and Ole e 1, major allergens from peanut and olive pollen, respectively, contained mainly xylosylated N-glycans with the composition Man(3(-4))XylGlcNAc(2) in the case of Ara h 1 and GlcNAc(1-2)Man(3)XylGlcNAc(2) in the case of Ole e 1 where also some GlcNAc(0-2)Man(3)XylFucGlcNAc(2) was found.

Allergens↗
PubMed2000

Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes.

Enzymatic formation of glutamate is critical to numerous biological pathways. However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery. We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases. This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M. Zhou, Z. Diwu, N. Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162-168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kinetic constants are comparable to literature values determined using a variety of assay techniques.

Glutamic Acid↗
PubMed2002

A fluorescence-based, high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.

Animals↗
PubMed1978

Iron hydroxide: model for enzymes that oxidize proteins.

The particles of an iron hydroxide sol were found to be a suitable model for protein-oxidizing enzymes such as peroxidase and polyphenol oxidase. In addition to small molecules such as pyrogallol, human serum proteins, albumin and gamma-globulin, are shown to be substrates of the oxidizing model. The activity is markedly increased by the addition of small amounts of copper to the iron in the particles of the sol. The size and molecular weight of the enzyme model, as well as the number of active centers were determined.

Blood Proteins↗
PubMed2002

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme.

The ability of a Brazilian strain of Pleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase), P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorization in vivo was tested using three dyes--Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability of P. pulmonarius to decolorize industrial dyes.

Brazil↗
PubMed1979

Biochemical and ultrastructural study of the disruption of blood platelets by streptolysin O.

The membrane-damaging protein toxin, streptolysin O, proved highly lytic on human, guinea-pig and rabbit platelets. About 15 molecules of toxin were sufficient to lyse one cell. Platelet disruption was assessed by electron microscopy, clearing of cell suspensions and assay of lactate dehydrogenase, serotonin, monoamine oxidase and glutathione peroxidase released in the extracellular fluid. This egress reflected the damage of both plasmic and organelle membranes. A quantitative study of lactate dehydrogenase and serotonin liberation taken as respective markers of the cytosol and dense bodies was undertaken as a function of toxin concentration. No platelet aggregation or shape change was elicited by streptolysin O. The ghosts resulting from platelet lysis retained properties of the native membrane such as aggregability and serotonin uptake. Dense bodies were easily separated after gentle disruption of the plasmic membrane by small amounts of toxin. Platelet lysis by streptolysin O proved a useful procedure for the determination of protein content, enzyme activities and serotonin assay on the same lysate in contrast to usual methods.

Animals↗
PubMed1986

Degradation of oxalate in rats implanted with immobilized oxalate oxidase.

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.

Animals↗