Showing papers on "Enzyme assay published in 1997"

The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland.

Abstract: A remarkably constant feature of vertebrate physiology is a daily rhythm of melatonin in the circulation, which serves as the hormonal signal of the daily light/dark cycle: melatonin levels are always elevated at night. The biochemical basis of this hormonal rhythm is one of the enzymes involved in melatonin synthesis in the pineal gland-the melatonin rhythm-generating enzyme-serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, E.C. 2.3.1.87). In all vertebrates, enzyme activity is high at night. This reflects the influences of internal circadian clocks and of light. The dynamics of this enzyme are remarkable. The magnitude of the nocturnal increase in enzyme activity ranges from 7- to 150-fold on a species-to-species basis among vertebrates. In all cases the nocturnal levels of AA-NAT activity decrease very rapidly following exposure to light. A major advance in the study of the molecular basis of these changes was the cloning of cDNA encoding the enzyme. This has resulted in rapid progress in our understanding of the biology and structure of AA-NAT and how it is regulated. Several constant features of this enzyme have become apparent, including structural features, tissue distribution, and a close association of enzyme activity and protein. However, some remarkable differences among species in the molecular mechanisms involved in regulating the enzyme have been discovered. In sheep, AA-NAT mRNA levels show relatively little change over a 24-hour period and changes in AA-NAT activity are primarily regulated at the protein level. In the rat, AA-NAT is also regulated at a protein level; however, in addition, AA-NAT mRNA levels exhibit a 150-fold rhythm, which reflects cyclic AMP-dependent regulation of expression of the AA-NAT gene. In the chicken, cyclic AMP acts primarily at the protein level and a rhythm in AA-NAT mRNA is driven by a noncyclic AMP-dependent mechanism linked to the clock within the pineal gland. Finally, in the trout, AA-NAT mRNA levels show little change and activity is regulated by light acting directly on the pineal gland. The variety of mechanisms that have evolved among vertebrates to achieve the same goal-a rhythm in melatonin-underlines the important role melatonin plays as the hormonal signal of environmental lighting in vertebrates.

Microchip Device for Performing Enzyme Assays

Abstract: An automated enzyme assay was performed within a microfabricated channel network. Precise concentrations of substrate, enzyme, and inhibitor were mixed in nanoliter volumes using electrokinetic flow. Reagent dilution and mixing were controlled by regulating the applied potential at the terminus of each channel, using voltages derived from an equivalent circuit model of the microchip. The enzyme β-galactosidase (β-Gal) was assayed using resorufin β-d-galactopyranoside (RBG), a substrate that is hydrolyzed to resorufin, a fluorescent product. Reaction kinetics were obtained by varying the concentration of substrate on-chip and monitoring the production of resorufin using laser-induced fluorescence. Derived Michaelis−Menten constants compared well between an on-chip and a conventional enzyme assay. Bias in the derived Km and kcat was primarily due to the limited solubility of RBG and the associated lack of measurements at substrate concentrations exceeding the Km. A Ki of 8 μM for the inhibitor phenylethyl β...

Inhibition of Cytochromes P-450 and Induction of Glutathione S-Transferases by Sulforaphane in Primary Human and Rat Hepatocytes

Abstract: The isothiocyanate sulforaphane (SF) is thought to be a potential chemoprotective agent. Its effects on Phase I and Phase II enzymes of carcinogen metabolism in primary cultures of rat and human hepatocytes have been investigated. Northern blot analyses of rat hepatocytes showed a dose-dependent induction of mRNAs for rat glutathione S-transferases (rGSTs) A1/A2 and P1 but not M1. This was associated with enhanced levels of not only rGSTA1, A2, A4, A5, and P1 but also of rGSTs M1 and M2. On the other hand, the enzyme activities in rat hepatocytes associated with cytochromes P-450 (CYPs) 1A1 and 2B1/2, namely ethoxyresorufin-O-deethylase and pentoxyresorufin-O-dealkylase, respectively, were decreased in a dose-dependent manner. In SF-treated human hepatocytes, hGSTA1/2 but not hGSTM1 mRNAs were induced, and the expression of CYP1A2 was unaffected, whereas the expression of CYP3A4, the major CYP in human liver, was markedly decreased at both mRNA and activity levels. These observations demonstrate that in intact human and rat hepatocytes, SF may both induce a number of GSTs and cause enzyme inhibition of some but not all CYPs and, in the case of CYP3A4, inhibit both its enzyme activity and its expression.

The influence of oxygen tension and pH on the expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast tumor cells grown in vitro and in vivo.

Abstract: We report that hypoxia regulates and influences the level of the angiogenic enzyme platelet-derived endothelial cell growth factor (PD-ECGF), also called thymidine phosphorylase, in vitro and in vivo. Levels of PD-ECGF protein increased 6-fold in the breast cancer cell line MDA 231 after 16 h of growth in 0.3% oxygen. A simultaneous increase in enzyme activity was observed. Immunohistochemical staining of MDA 231 tumors grown in nu/nu mice showed increased expression of PD-ECGF in those parts of the tumor that are proximal to the areas of necrosis. In addition, increased and widespread staining for PD-ECGF protein was obtained when the tumor vascular supply was occluded for 2 h by clamping. Lowering the media pH to 6.3-6.7 in vitro also resulted in an increase in PD-ECGF protein levels. This study demonstrates that tumor microenvironmental factors can result in the specific up-regulation of an angiogenic enzyme that can also activate 5-fluorouracil prodrugs and hence is exploitable therapeutically.

Localization and activity of lysyl oxidase within nuclei of fibrogenic cells

Abstract: Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages.

Abstract: Activation of rat peritoneal macrophages by LPS resulted in time-dependent production of nitric oxide and enhancement of cyclooxygenase (Cox) activity. This stimulation was accompanied by increased expression of inducible enzymes, NO synthase, and Cox-2, contrasting with no variation in constitutive Cox-1. Inhibition of NO production in LPS-treated macrophages by either the L-arginine analogue N(G)-monomethyl-L-arginine (L-NMMA) or aminoguanidine was accompanied by an additional enhancement of Cox activity parallel to the expression of Cox-2 protein analyzed by Western blot. Addition of NO donors (3-morpholinosydnonimine (Sin-1), sodium nitroprusside, or S-nitrosoglutathione) reversed the effects of L-NMMA, confirming the role of NO on Cox-2 expression. Specific immunoprecipitation of Cox-2 showed a pattern of protein expression similar to that observed in intact cells. Enzyme activity tested on the immunoprecipitates was correlated with the enzyme mass. In contrast, there was no variation in immunoprecipitated Cox-1 protein or in activity. Levels of mRNA for Cox-2 were increased in macrophages stimulated by LPS in the presence of L-NMMA compared with LPS alone. Metabolic labeling using [35S]methionine showed that inhibition of NO formation resulted in enhanced de novo synthesis of the 35S-labeled Cox-2. The amount of Cox-2 protein induced in the presence or absence of L-NMMA did not change for at least 6 h, suggesting that NO does not modify the t1/2 of the enzyme. These results provide evidence that NO participates in PG production by negative regulation of Cox-2 expression.

Characterization of phytochelatin synthase from tomato

Abstract: The enzyme that synthesizes Cd-binding phytochelatins (PCs), PC synthase, has been studied in tomato (Lycopersicon esculentum) cell cultures and plants. This enzyme transfers γ-GluCys from GSH or PC to either GSH or an existing polymer of (γ-GluCys) n Gly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ-GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd 2+ , Ag + . Cu 2+ , Au + , Zn 2+ , Fe 2+ , Hg 2+ and Pb 2+ . In crude protein extracts from tomato cells the enzyme has an apparent K m of 7.7 mM for GSH in the presence of 0.5 mM Cd 2+ , and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium-resistant tomato cells growing in medium containing 6 mM CdCl 2 have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd-PC complexes in response to cadmium.

Identification of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida flavanone 3beta-hydroxylase.

Abstract: Flavanone 3β-hydroxylase, involved in the biosynthesis of flavonoids, catechins, and anthocyanidins, is a non-heme iron enzyme, dependent on Fe2+, molecular oxygen, 2-oxoglutarate, and ascorbate, the typical cofactors of the class of 2-oxoglutarate-dependent dioxygenases. Sequence alignment analysis of various 2-oxoglutarate-dependent dioxygenases and related enzymes revealed eight amino acid residues that seem to be strictly conserved within this group of enzymes. Among these residues, two histidines (His220 and His278) and one aspartic acid (Asp222) were identified as part of the putative iron-binding site and an arginine residue (Arg288) as part of the 2-oxoglutarate binding site, by site-directed mutagenesis and functional analysis of the mutated recombinant enzyme. The mutant genes were expressed in Escherichia coli to give soluble proteins whose molecular masses were in excellent agreement with the wild-type enzyme. Four out of nine mutant enzymes, [Gln78]FHT, [Gln121]FHT, [Gln264]FHT and [Gln266]FHT, were enzymatically active with activities reduced to 26–57%, implying that the mutated amino acid residues are not essential for catalysis. Replacement of His220 by glutamine and Asp222 by asparagine remarkably reduced the catalytic activity to about 0.15% and 0.4%, respectively. The [Gln220]FHT and [Asn222]FHT enzymes showed a slightly increased Km value with respect to iron binding, as compared to the wild-type enzyme. The most drastic effect on the reaction rate of flavanone 3β-hydroxylase was achieved by mutating His278 to glutamine. The mutant had no detectable enzyme activity, indicating that His278 was essential for the catalytic reaction. The observed protection of purified enzyme from inactivation by diethylpyrocarbonate after the addition of cofactors provided further independent confirmation for the involvement of histidine residues in the active site. The substitution of Arg288 by lysine or glutamine induced a precipitous decrease in catalytic activity and a fivefold and 160-fold increase in the Michaelis constants for 2-oxoglutarate, respectively. In addition, the enzymatic activities of the latter two mutant enzymes showed a strong pH dependence in the weakly acidic as well as in the neutral pH range, unlike the wild-type enzyme. These results clearly indicate that Arg288 probably contributes to the specific binding of 2-oxoglutarate at the active site of the enzyme, most likely by providing a positive charge, properly located in order to interact with the (5-carboxyl function of 2-oxoglutarate. Furthermore, we conclude that His220, His278 and Asp222 constitute three of the possible ligands for iron binding in the active site of flavanone 3β-hydroxylase.

Effect of cadmium on proline accumulation and ribonuclease activity in rice seedlings: role of proline as a possible enzyme protectant

Abstract: When seedlings of two rice (Oryza sativa L) cultivars Ratna and Jaya were raised under 100 and 500 µM cadmium nitrate in the medium, a high proline content was noted in Cd2+ stressed seedlings compared to controls Seedlings grown under 500 µM Cd(NO3)2 maintained increased proline level compared to non-stressed seedlings Kinetic properties of RNase extracted from control grown and Cd2+ stressed seedlings showed a marked alteration in Km due to Cd2+ treatment The RNase isoforms were purified from 15-d-old rice seedlings with a total purification of 2225 fold and 7475 % yield using conventional biochemical techniques Three RNase isoforms, namely I, II and III were eluted from DEAE-Sephacel column The isoform RNase II had Km value of 32 mg(RNA) cm-3 The in vitro osmotic stress created by incorporation of PEG in the enzyme assay medium led to decreased affinity of enzyme towards its substrate with increase in Km This loss in affinity was partially restored by the addition of 1 M proline in the assay medium, suggesting the possible protective role of proline on RNase under osmotic stress

Molecular Cloning of 4-Coumarate:Coenzyme A Ligase in Loblolly Pine and the Roles of This Enzyme in the Biosynthesis of Lignin in Compression Wood

Abstract: Two genomic sequences encoding 4-coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) in loblolly pine (Pinus taeda L.) were cloned. Both sequences contained three introns and four exons with identical coding sequences predicting 537 amino acids. Two of the three introns in these two clones were different both in sequence and in length. Sequences of both 4CL clones were found in all nine megagametophyte DNAs tested, providing genetic evidence that these two 4CL genomic sequences are nonallelic genes. Our analyses suggest that there are at least two distinct, intron-containing 4CL genes, at least one of which is transcribed into 4CL mRNA in developing xylem tissue of loblolly pine. The levels of 4CL gene transcription in xylem were influenced by compressional stress, resulting in an elevated 4CL enzyme activity with 4-coumaric acid. 4CL enzyme activity with ferulic acid remained unchanged, whereas with caffeic acid it was significantly inhibited. Exogenously applied trans-cinnamic acid in the protein extracts from normal wood xylem caused inhibition of 4CL activity toward caffeic acid similar to that under compressional stress. The implications of this cinnamic acid-modulated effect on 4CL enzyme activities toward different substrates in regulating monolignol synthesis in xylem under compressional stress are discussed.

Relationship between the Glutamate Production and the Activity of 2-Oxoglutarate Dehydrogenase in Brevibacterium lactofermentum

Abstract: Enzyme activities of 2-oxoglutarate dehydrogenase complex and glutamate dehydrogenase of wild type Brevibacterium lactofermentum, one of the typical glutamate-producing coryneform bacteria, were investigated by using cells cultured under glutamate-productive and glutamate-non-productive conditions. Significant reduction of the former enzyme activity was observed in the cells under the several glutamate-productive conditions, namely, in the cells cultured in media containing a) limited concentrations of biotin, b) sub-lethal amounts of penicillin, and c) sub-optimal amounts of a surface-active agent, as compared with those under the non-productive conditions. The activity of the latter enzyme was essentially unchanged in every condition. The relationship between glutamate production and the enzyme activities as well as permeability of glutamate through cell membrane was discussed from the results obtained.

Genetic Manipulation of Key Photosynthetic Enzymes in the C4 Plant Flaveria bidentis

Abstract: The NADP-malic enzyme type C 4 dicot Flaveria bidentis (L.) Kuntze was transformed with antisense and cosense gene constructs that resulted in specific decreases in single photosynthetic enzymes. The enzymes targeted were ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (Rubisco), pyruvate, Pi dikinase (EC 2.7.9.1) (PPDK) and NADP malate dehydrogenase (EC 1.1.1.82) (NADP-MDH). These enzymes were chosen as targets because they have low activity compared to photosynthetic rates (Rubisco), are subject to complex covalent regulation (NADP-MDH), or both (PPDK). T1 progeny of a number of lines of these transformants were examined for the effects of these gene constructs on enzyme levels and photosynthetic performance. Rubisco antisense transformants expressing between 15 and 100% of wild-type enzyme activity were obtained. Pyruvate, Pi dikinase antisense lines were obtained with 40Ð100% wild-type levels. NADP malate dehydrogenase sense constructs caused a co-suppression of enzyme activity with some lines containing less than 2% of wild- type activity. Under saturating illumination, the control coefficients for these enzymes were determined to be up to 0.7 for Rubisco, 0.2Ð0.3 for PPDK and effectively zero for NADP-MDH. The implications of these observations for the regulation of photosynthetic flux and metabolism in C 4 plants and the role of regulation by covalent modification are discussed.

Metabolism of Naphthalene, 1-Naphthol, Indene, and Indole by Rhodococcus sp. Strain NCIMB 12038.

Abstract: The regulation of naphthalene and 1-naphthol metabolism in a Rhodococcus sp. (NCIMB 12038) has been investigated. The microorganism utilizes separate pathways for the degradation of these compounds, and they are regulated independently. Naphthalene metabolism was inducible, but not by salicylate, and 1-naphthol metabolism, although constitutive, was also repressed during growth on salicylate. The biochemistry of naphthalene degradation in this strain was otherwise identical to that found in Pseudomonas putida, with salicylate as a central metabolite and naphthalene initially being oxidized via a naphthalene dioxygenase enzyme to cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene (naphthalene cis-diol). A dioxygenase enzyme was not expressed under growth conditions which facilitate 1-naphthol degradation. However, biotransformations with indene as a substrate suggested that a monooxygenase enzyme may be involved in the degradation of this compound. Indole was transformed to indigo by both naphthalene-grown NCIMB 12038 and by cells grown in the absence of an inducer. Therefore, the presence of a naphthalene dioxygenase enzyme activity was not necessary for this reaction. Thus, the biotransformation of indole to indigo may be facilitated by another type of enzyme (possibly a monooxygenase) in this organism.

Influence of Gene Dosage on Carbohydrate Synthesis and Enzymatic Activities in Endosperm of Starch-Deficient Mutants of Maize

Abstract: In cereals, starch is synthesized in endosperm cells, which have a ploidy level of three. By studying the allelic dosage of mutants affecting starch formation in maize (Zea mays L.) kernels, we determined the effect of down-regulated enzyme activity on starch accumulation and the activity of associated enzymes of carbohydrate metabolism. We found a direct relationship between the amount of starch produced in the endosperm and the gene dosage of amylose extender-1, brittle-2, shrunken1, and sugary-1 mutant alleles. Changes in starch content were found to be caused by changes in the duration as well as the rate of starch synthesis, depending on the mutant. Branching enzyme, ADP-glucose pyrophosphorylase, and sucrose synthase activities were linearly reduced in endosperm containing increasing dosages of amylose extender-1, brittle-2, and shrunken-1 alleles, respectively. De-branching enzyme activity declined only in the presence of two or three copies of sugary-1. No enzyme-dosage relationship occurred with the dull1 mutant allele. All mutants except sugary-1 displayed large increases (approximately 2- to 5-fold) in activity among various enzymes unrelated to the structural gene. This occurred in homozygous recessive genotypes, as did elevated concentrations of soluble sugars, and differed in magnitude and distribution among enzymes according to the particular mutation.

Reagentless mediated laccase electrode for the detection of enzyme modulators.

Abstract: We have investigated aerobic mediation of electron transfer to a laccase enzyme by the solution redox couples [Os(bpy)2Cl2]+/0 and [Os(bpy)2(MeIm)Cl]2+/+, where bpy is 2,2-bipyridine and MeIm is N-methylimidazole. The factors that influence the homogeneous mediation reaction are investigated and discussed. Investigation of ionic strength, pH, and temperature effects on the kinetics of intermolecular electron transfer elucidates the governing factors in the mediator−enzyme reactions. Coimmobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the reagentless addressing of enzyme activity, consuming only oxygen present in solution. Thus, these immobilized enzyme biosensors can be utilized for the detection of modulators of laccase activity, such as the inhibitor sodium azide. The enzyme inhibition biosensor can detect levels of azide as low as 2.5 × 10-6 mol dm-3 in solution.

Conversion of dihydroceramide into ceramide: involvement of a desaturase

Abstract: Ceramide has been suggested to be a potent bioactive lipid involved in cell growth, differentiation and apoptosis. Its precursor, dihydroceramide, does not affect these processes. The truncated dihydroceramide analogues N-hexanoyl-[4,5-H-3]-D-erythro-sphinganine and N-[1-C-14]-hexanoyl-D-erythro-sphinganine were used to study the conversion of dihydroceramide into ceramide by rat hepatocytes. The formation of tritiated water after the addition of the tritiated substrate to intact and permeabilized rat hepatocytes was followed to measure enzyme activity. Desaturation was severely depressed in permeabilized hepatocytes, suggesting loss of cofactors. Of a variety of cofactors tested in the permeabilized cells, NADPH appeared to be stimulatory, pointing to the involvement of a desaturase. In agreement with this, the addition of inhibitors and redox effecters known to affect Delta(9)-stearoyl-CoA desaturase and Delta(1)-plasmanyl-ethanolamine desaturase to intact cells resulted in severe inhibition of the desaturation. When added to permeabilized cells fortified with NADPH, these compounds counteracted the NADPH stimulation. The enzyme system was further studied in broken cells. On cell fractionation, the activity was recovered in the microsomal fraction. The results indicate that the conversion of dihydroceramide into ceramide is catalysed by a desaturase and not by a dehydrogenase or an oxidase as was generally believed.

Involvement of two alpha-ketoglutarate-dependent dioxygenases in enantioselective degradation of (R)- and (S)-mecoprop by Sphingomonas herbicidovorans MH.

Abstract: Cell extracts of Sphingomonas herbicidovorans MH grown on (R)-mecoprop contained an enzyme activity that selectively converted (R)-mecoprop to 4-chloro-2-methylphenol, whereas extracts of cells grown on (S)-mecoprop contained an enzyme activity selective for the S enantiomer. Both reactions were dependent on alpha-ketoglutarate and ferrous ions. Besides 4-chloro-2-methylphenol, pyruvate and succinate were detected as products of the reactions. Labeling experiments with (18)O2 revealed that both enzyme activities catalyzed a dioxygenation reaction. One of the oxygen atoms of pyruvate and one of the oxygen atoms of succinate were derived from molecular oxygen. Analysis of cell extracts obtained from cells grown on different substrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that growth on (R)-mecoprop and (S)-mecoprop caused the appearance of prominent protein bands at 34 and 32 kDa, respectively. Both protein bands were present when cells grew on the racemic mixture. The results demonstrate that S. herbicidovorans initiated the degradation of each enantiomer of mecoprop by a specific alpha-ketoglutarate-dependent dioxygenase. By comparing conversion rates of various phenoxy herbicides, we confirmed that the two enzyme activities were distinct from that of TfdA, which catalyzes the first step in the degradation of 2,4-dichlorophenoxyacetic acid in Ralstonia eutropha JMP134.