A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity

A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.     

A continuous spectrophotometric assay for the determination of diamondback moth esterase activity

Conventional methods to determine esterase activity from insects are composed of a three-step process where the enzyme is allowed to hydrolyze a 1-naphthyl acetate substrate, that reaction is quenched by a SDS detergent, and then a Fast Blue B dye complex is formed with 1-naphthol, the product of 1-naphthyl acetate hydrolysis. These methods measure dye-product complex rather than the product, 1-naphthol. A new assay is presented that continuously monitors the formation of 1-naphthol with the hydrolysis of an esterase substrate. The esterase activity was determined as the slope of the linear regression change in absorbance over time at 320 nm. The continuous assay provides a simple, rapid, and sensitive method for measuring esterases extracted from a single diamondback moth in 1-10 min. The detection limit of the assay is approximately 0.6 microM 1-naphthol. The 1-naphthol product from the esterase reaction was confirmed by HPLC analysis. According to the assay, the K(m) and V(max) values of the esterase were 28 +/- 2 microM and 6.0 +/- 0.1 microM/min, respectively, at 37 degrees C for 1-naphthyl acetate. The K(i) value was 9 +/- 2 microM using azadirachtin, an insecticide from neem tree, Azadirachta indica (A.Juss). Azadirachtin was a reversible competitive inhibitor of the esterase activity.     

A new continuous spectrophotometric assay method for DOPA oxidase activity of tyrosinase

Sensitive assay methods for tyrosinase are essential not only for the understanding the process of pigment production but also for the development of effective inhibitors of tyrosinase. To develop an efficient assay method, we applied thymol blue to reaction mixtures. The enzyme kinetic study revealed that DOPA oxidase activity of tyrosinase in thymol blue-applied reaction system was more sensitively measured, even under lower enzyme units compared with the previous report with significant enhancement of Vmax while affinity change on substrate was not observed. To test whether this method could be applicable to the inhibition and the inactivation kinetic study of tyrosinase, the effect of kojic acid, a well-known tyrosinase inhibitor, and sodium chloride respectively, have been studied. Conclusively, thymol blue method can assay tyrosinase activity with sensitivity and is applicable to the inhibition and the inactivation study of tyrosinase.     

A new method for spectrophotometric assay of activity of cross-linked penicillin acylase aggregates

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability of immobilized enzyme preparations.     

An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels

The use of 3,3′,5,5′-tetramethylbenzidine-H₂O₂ as a stain for the peroxidase activity of cytochrome P-450 (or cytochrome P-450 in sodium dodecyl sulfate polyacrylamide gels is described in this report. This reagent can be used to detect very low levels of heme-associated peroxidase activity. The blue-stained bands on polyacrylamide gels are distinet, and the color is stable. The stained gels can be photographed or scanned at 690 nm because the gel background remains clear. The stain is easily removed from the gels to permit subsequent protein staining. Staining first for peroxidase activity has no effect on the subsequent protein staining profile. The peroxidase activity of cytochrome P-450 (or cytochrome P-420) in immunoprecipitates in Ouchterlony double diffusion plates can also be detected using this reagent.     

A spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2: comparison with fluorometric assay

A simple spectrophotometric method to monitor the catalytic activity of microsomal cytochrome P-450 IIB1/2 has been developed. The method employs measurement of utilization of NADPH, consumption of the substrate, pentoxyresorufin (PRF) and formation of the product, resorufin (RF) in the same reaction mixture containing hepatic microsomes from phenobarbital treated rats. The velocity of NADPH utilization (16.36 nmole/min/nmole P-450), PRF consumption (1.58 nmole/min/nmole P-450) and RF formation (1.57 nmole/min/nmole P-450) suggested a stoichiometry of 1:1 between the substrate and the product alongwith utilization of 10 molecules of NADPH. However, the Km for the enzyme activity (nmole RF formed/min/nmole P-450) using varying concentrations of PRF and NADPH as substrates were found to be 11.6 and 20.2 microM, respectively. The spectrophotometric method was compared with fluorometric method in terms of linearity with time, P-450 content and Vmax, Km values observed for the reaction. Inhibition studies with metyrapone and SKF 525A in the utilization of NADPH, consumption of PRF and formation of RF suggested that the method could be useful in monitoring the effect of various inhibitors on the P-450 IIB1/2 reaction.     

Differential action of thyroid hormones and chemically related compounds on the activity of UDP-glucuronosyltransferases and cytochrome P-450 isozymes in rat liver

The effect of thyroid hormones and chemically related compounds, on the activity of UDP-glucuronosyltransferases (EC 2.4.1.17) and cytochrome P-450-dependent monooxygenases in rat liver microsomes was investigated. The animals were thyroidectomized and treated with different doses of the drugs for 3 weeks. Opposite effects were observed depending on the isoenzyme of UDP-glucuronosyltransferase considered. While 3,3′,5-triiodo-l-thyronine, 3,3′,5-triiodothyroacetic acid, 3,3′,5-triiodothyropropionic acid, isopropyldiiodothyronine and l- and d-thryoxine strongly increased 4-nitrophenol glucuronidation in a dose-dependent fashion, they decreased markedly bilirubin glucuronidation. However, the activity toward nopol, a monoterpenoid alcohol, was not significantly changed regardless of which compound or dose was used. Variation of UDP-glucuronosyltransferase observed with 4-nitrophenol and bilirubin was related to the thyromimetic effect of the drugs estimated from the increase in α-glycerophosphate dehydrogenase. Thyronine and 3,5-diiodo-l-tyrosine, which did not enhance this activity, also failed to affect glucuronidation. Variations in UDP-glucuronosyltransferase activity were more likely due to changes in protein expression rather than changes in enzyme latency, since lipid organization of the microsomal membrane, as estimated from the mean anisotropy of 1,6-diphenyl-1,3,5-hexatriene by fluorescence polarization was not significantly modified by the drug administration. Although some of the drugs could significantly decrease the triacylglycerol and cholesterol contents in plasma, all failed to affect lauric acid hydroxylation. The activities of catalase, palmitoyl-CoA dehydrogenase (CN^? insensitive) and carnitine acetyltransferase in the fraction enriched in peroxisomes were also not significantly affected by treatment with the thyroid hormone LT₃. In contrast, the activity of 7-ethoxycoumarine O-deethylase was increased by large doses of thyronine and by 3,3′,5′-triiodothyropropionic acid. The concentration of total cytochrome P-450 was decreased in a dose-dependent fashion by all the compounds used, except thyronine. Finally, significant correlations were observed between glucuronidation of bilirubin and 4-nitrophenol and the content in cytochrome P-450. This suggests a possible coordinate regulation of the two processes, which depends on the physicochemical characteristics of the thyroid hormones and related compounds.     

A limitation of the continuous spectrophotometric assay for the measurement of myo-inositol-1-phosphate synthase activity

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the conversion of glucose-6-phosphate to myo-inositol-1-phosphate. The reaction catalyzed by MIPS is the first step in the biosynthesis of inositol and inositol-containing molecules that serve important roles in both eukaryotes and prokaryotes. Consequently, MIPS is a target for the development of therapeutic agents for the treatment of infectious diseases and bipolar disorder. We recently reported a continuous spectrophotometric method for measuring MIPS activity using a coupled assay that allows the rapid characterization of MIPS in a multiwell plate format. Here we validate the continuous assay as a high-throughput alternative for measuring MIPS activity and report on one limitation of this assay—the inability to examine the effect of divalent metal ions (at high concentrations) on MIPS activity. In addition, we demonstrate that the activity of MIPS from Arabidopsis thaliana is moderately enhanced by the addition Mg²⁺ and is not enhanced by other divalent metal ions (Zn²⁺ and Mn²⁺), consistent with what has been observed for other eukaryotic MIPS enzymes. Our findings suggest that the continuous assay is better suited for characterizing eukaryotic MIPS enzymes that require monovalent cations as cofactors than for characterizing bacterial or archeal MIPS enzymes that require divalent metal ions as cofactors.     

A novel assay system for the measurement of transketolase activity using xylulokinase from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The conventional method of transketolase (TKT) activity assay uses ribose 5-phosphate and xylulose 5-phosphate as substrates. However, a new method of TKT assay is currently required since xylulose 5-phosphate is no longer commercially available and is difficult to synthesize chemically. Although there are effective assays for TKT using non-natural substrates, these are inadequate for evaluating changes in enzyme activity and affinity toward real substrates. As a solution to such problems, we describe a novel assay system using xylulokinase (XK) from Saccharomyces cerevisiae. As for this purpose, the XK was overexpressed in E. coli, separated and purified in a single step, added to induce a reaction that generated xylulose 5-phosphate, which was integrated into the conventional TKT assay. The new coupling assay gave reproducible results with E. coli TKT and had a detection limit up to 5 × 10⁻⁴ unit/mg protein. A reliable result was also achieved for the incorporation of XK and TKT into a single reaction.     

Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: A novel assay for dihydropteroate synthase

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPP_i to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/α-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.     

A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

A method for activity staining after native polyacrylamide gel electrophoresis using a coupled enzyme assay and fluorescence detection: application to the analysis of several glycolytic enzymes.

We describe a method for the detection of isoforms of several glycolytic enzymes by activity staining after native PAGE. The staining is based on coupled enzyme assays carried out on the gel after electrophoresis and is linked to the disappearance of NADH, which is visualized by fluorescence. This method offers reliable and sensitive detection for phosphoenolpyruvate carboxylase, PPi-dependent phosphofructokinase, and pyruvate kinase from plant tissues. It can be applied to the detection of all enzymes which are normally detected spectrophotometrically using coupled enzyme assays consuming NAD(P)H.     

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.     

A fast and efficient method for quantitative measurement of S-adenosyl-l-methionine-dependent methyltransferase activity with protein substrates

Modification of protein residues by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1 h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.     

Extractionless method for the simultaneous high-performance liquid chromatographic determination of urinary caffeine metabolites for N-acetyltransferase 2, cytochrome P450 1A2 and xanthine oxidase activity assessment

Urinary metabolic ratios of caffeine are used in humans to assess the enzymatic activities of cytochrome P450 isoenzyme 1A2 (CYP1A2), xanthine oxidase (XO) and for phenotyping individuals for the bimodal N-acetyltransferase 2 (NAT2), all of them involved in the activation or detoxification of various xenobiotic compounds. Most reported analytical procedures for the measurement of the urinary metabolites of caffeine include a liquid–liquid extraction of urine samples prior to their analysis by reversed-phase HPLC. At neutral to basic pH however, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), a metabolite of caffeine, spontaneously decomposes to 5-acetylamino-6-amino-3-methyluracil (AAMU). Since AAMU is not extracted in most organic solvents, the extent of AFMU decomposition cannot be precisely assessed. Although the decomposition reaction can be minimized by immediate acidification of the urine, accurate results can only be obtained when both AAMU and AFMU are monitored, or alternatively, if AAMU is measured after complete transformation of AFMU into AAMU in basic conditions. We report a liquid chromatographic method for the simultaneous quantitative analysis of the five urinary metabolites of caffeine used for the CYP1A2, XO and NAT2 phenotyping studies: AAMU, AFMU, 1-methylxanthine, 1-methyluric acid and 1,7-dimethyluric acid. These metabolites are satisfactory separated from all other known caffeine metabolites as well as endogenous urinary constituents. Sample treatment does not require any liquid–liquid extraction procedure. Urine samples are diluted and centrifuged before being injected (10 μl) onto a YMC-Pack Polyamine II (250×4.6 mm) column. A step-wise gradient elution program is applied using acetonitrile–0.75% (v/v) formic acid: (91:9) at 0 min→(75:25) at 25 min→(65:35) at 35 min→(65:35) at 45 min, followed by a re-equilibration step to the initial solvent composition. The flow-rate is 1.0 ml/min and the separations are monitored by UV absorbance at 260 and 280 nm. The procedure described here represents a substantial improvement over previous methods: a single analysis and a minimal urine sample treatment enables the simultaneous quantitation of five caffeine metabolites, notably AFMU and AAMU, used for the determination of CYP450 1A2, XO and NAT2 enzyme activity. Importantly enough, phenotyping individuals for the bimodal NAT2 is made possible without the uncertainty associated with the deformylation of AFMU, which is likely to happen at all steps prior to the analysis, during sample storage and even in the bladder of the subjects.