A radioactive assay for guanidoacetate methyltransferase.

A radioactive assay for guanidoacetate methyltransferase is described. It is based on a separation by an AG 50 (NH₄⁺) column, on which [methyl-¹⁴C]Adenosylmethionine is adsorbed, in contrast to [methyl-¹⁴C]creatine which can be collected in the effluent fraction. Guanidoacetate methyltransferase is sensitive to inhibition by adenosylhomocysteine and 3-deazaadenosylhomocysteine.     

A simple heterogeneous one-step assay for screening estrogenic compounds

Estrogen receptor (ER) modulators are a serious health issue but estrogenic compounds, especially antagonists of ER function, are widely screened for in search of novel therapeutics against hormonal diseases such as the breast cancer. Here we report a novel and a simple bioassay for estrogenic and anti-estrogenic compounds based on ligand-dependent recruitment of ER co-activator steroid receptor co-activator 1 (SRC-1) to purified Renilla luciferase-tagged ERα. In this assay, in vivo-biotinylated (E. coli) SRC-1, purified Renilla luciferase-ERα, and the analyte sample are mixed and incubated for 2 h in a streptavidin-coated microtiter wells, and after one washing step, luminescence is measured with a simple instrument. The assay does not require chemical labeling of the components and shows good sensitivity (25 pM E₂) and wide dynamic range of more than four orders of magnitude.     

A nuclear-magnetic-resonance-based assay for betaine-homocysteine methyltransferase activity

Betaine-homocysteine methyltransferase (BHMT) activity can be measured directly and kinetically by (1)H-nuclear magnetic resonance spectroscopy. The disappearance of substrates and the formation of products are monitored simultaneously. Alternative substrates, separately and when mixed with glycine betaine, can also be monitored. Each assay can be completed in 1h. Using 2mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH 7.5) and measuring the production of N,N-dimethylglycine, the CV is 6.3% (n=6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed, by measuring the N,N-dimethylglycine produced after incubation with 2 mM glycine betaine and homocysteine (CV=5.3%, n = 6) with a detection limit of 2 nkatal. These assays were used to show that the natural betaines trigonelline, proline betaine, arsenobetaine, and l-carnitine are neither substrates nor significant inhibitors of rat liver BHMT, that the thetins dimethylthetin and dimethylsulfoniopropionate are substrates and inhibit methyl transfer from glycine betaine, and that the K(m) for glycine betaine is 0.19+/-0.03 mM with a V(max) of 17+/-0.7 nMol min(-1) mg(-1).     

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity

A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 ± 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R²) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5′-CG-3′ site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.     

A simple direct spectrophotometric assay for 24,25-dihydroxyvitamin D3

A simple reproducible assay to determine the concentration of 24,25-dihydroxyvitamin D₃ in blood plasma is described. This technique employs quantitation of the steroid by direct spectrophotometric analysis, rather than competitive protein binding assay after partition and high-pressure liquid chromatographic purification of plasma extracts. The concentration of metabolite observed in normal adult plasma is 2.4 ± 1.1 ng/ml.     

A direct continuous spectrophotometric assay for transglutaminase activity

Herein we report the development of a direct and continuous spectrophotometric method for determining transglutaminase (TGase) activity by using N,N-dimethyl-1,4-phenylenediamine (DMPDA) as a gamma-glutamyl acceptor substrate and carbobenzyloxy-l-glutamylglycine (Z-Gln-Gly) as a typical peptide gamma-glutamyl donor substrate. The transamidation activity of TGase can thus be followed by monitoring the increase of absorbance of the resulting anilide product at 278 nm. The extinction coefficient of the authentic, independently synthesized anilide was determined to be epsilon = 8940 +/- 55 M(-1) cm(-1). Using this assay, we determined the apparent K(M) of DMPDA to be 0.25 mM, which compares favorably to the apparent K(M) values determined for other acceptor substrates under conditions where Z-Gln-Gly is also used as the donor substrate, such as N-acetyl-l-lysine methyl ester (9.6 mM) and methylamine (13.1 mM). Finally, the sensitivity of this assay technique was established through the measurement of irreversible inhibition constants for iodoacetamide, determined to be K(I) = 75 +/- 11 nM and k(inact) = (120 +/- 1) x 10(5) M(-1) min(-1).     

A new method for the assay of glutaminase activity: direct measurement of product formation by an ammonia electrode

A simple, reliable procedure is described for the quantitative assay of glutaminase reaction by measuring product formation using an ammonia electrode. The ammonia electrode is a gas-detecting electrode, sensing the level of dissolved ammonia in aqueous solutions. Ammonia concentration can be read from calibration curves after converting ammonium ion to ammonia by adding sufficient base. Sample color and turbidity do not affect measurements, and samples need not be distilled. The concentrations of the three glutaminase isoenzymes from rat tissues measured by this method are strictly comparable to those measured by other methods.     

A bioluminescence assay for DNA methyltransferase activity based on methylation-resistant cleavage

DNA methyltransferase (MTase) is a kind of important regulatory factor in various biological processes. Current methods to investigate DNA MTase activity are still limited in the sensitivity and/or generality. Therefore, developing methods with high sensitivity and improved generality is needed. Here, we develop a new bioluminescence strategy based on methylation-resistant cleavage and protein expression in vitro to detect DNA MTase activity. In the strategy, Dam MTase was used as a model enzyme and MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) was used as their action target. Because the completely methylated LR-DNA could be expressed as detectable luciferase, Dam MTase activity was quantified by measuring the luminescence intensity of the expressed luciferase. The assay provides a very low detection limit (0.08 U/ml) as well as a wide linear range (0.2-100 U/ml). Besides, the analysis mode has improved generality and could be extended to the detection of other DNA MTases and the corresponding inhibitor screening.     

A simple assay for methionine adenosyltransferase using cation exchange paper and liquid scintillation spectrometry

A rapid, sensitive and simple procedure for the assay of l-methionine-S-adenosyltransferase based on the use of phosphocellulose ion-exchange paper is presented. The analytical procedure may be generally useful for all enzymes where there is a large cationic charge difference between the substrates and products. An application of a simpllified counting procedure for radioisotopes on solid supports is presented.     

A simple assay for detection of small-molecule redox activity

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts.     

A rapid assay for activity of phospholipase A2 using radioactive substrate

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.     

A simple spectrophotometric assay for urinary leukocyte esterase activity

We have developed a sensitive spectrophotometric method for assaying urinary leukocyte esterase activity by employing a synthetic substrate, N-toluene sulfonyl indoxyl alanine ester. This kinetic assay can be performed with a small aliquot of urine, by following the change in absorbance of the chromophore at 385 nm. It is rapid and specific for leukocyte esterase and therefore can be used in the early diagnosis of urinary tract infection.     

A simple radiometric assay for estradiol 2-hydroxylase activity

[2-³H]Estradiol was synthesized from 2-iodoestradiol by reduction with sodium borotritiride in the presence of palladium chloride as a catalyst. When the labeled substrate was incubated with the rat liver and kidney microsomes, the tritium label was liberated quantitatively depending upon the 2-hydroxylase activity. Tritiated water produced in the incubation medium was recovered as a satisfactory rate by passage through a column of Amberlite XAD-2 resin without any interference due to the labeled substrate. The present method for the assay of estradiol 2-hydroxylase activity was found to be simple, reliable, and sensitive (detection limit:29 pmol of 2-hydroxyestradiol).     

A simple one-step hemolytic assay for C2 with C2-deficient human serum.

A simple one-step procedure has been developed for the molecular titration of C2 by utilizing the ability of the test material to restore the hemolytic activity of human serum selectively deficient in C2 (C2D serum). In this assay, equal volumes of EA (10(8) cells/ml), C2D serum (1/20), and a suitable dilution of a source of C2 were incubated at 37 degrees C for 60 min and the fraction of cells lysed was used to calculate the effective molecules of C2/ml test material. The assay can be used to titrate C2 in human, guinea pig, rat, mouse and rabbit sera, but not C2 in dog serum. The assay is simple and reproducible, and comparable in sensitivity to the conventional two-step assay with EAC14 cells and Cgp-EDTA.     

A simple, one-step chromatographic procedure for the purification of lysozyme

A rapid method for the purification of lysozyme (mucopeptide N-acetyl-muramoylhydrolase, EC 3.2.1.17) from hen egg-white has been devised. It was that gel filtration chromatography on agarose columns can be used selectively to purify lysozyme, due to the fact that this protein interacts with the agarose matrix and elutes later than the corresponding total volume for the column. Thus, lysozyme is directly obtained in a relatively pure form and with a high specific activity. In principle, this simple method can be used to prepare lysozymes from other sources.     

A direct fluorescence-based assay for RGS domain GTPase accelerating activity

Diverse extracellular signals regulate seven transmembrane-spanning receptors to modulate cellular physiology. These receptors signal primarily through activation of heterotrimeric guanine nucleotide binding proteins (G proteins). A major determinant of heterotrimeric G protein signaling in vivo and in vitro is the intrinsic GTPase activity of the Galpha subunit. RGS (regulator of G protein signaling) domain-containing proteins are GTPase accelerating proteins specific for Galpha subunits. In this article, we describe the use of the ribose-conjugated fluorescent guanine nucleotide analog BODIPYFL-GTP as a spectroscopic probe to measure intrinsic and RGS protein-catalyzed nucleotide hydrolysis by Galphao. BODIPYFL-GTP bound to Galphao exhibits a 200% increase in fluorescence quantum yield. Hydrolysis of BODIPYFL-GTP to BODIPYFL-GDP reduces the quantum yield to 27% above its unbound value. We demonstrate that BODIPYFL-GTP can be used as a rapid real-time probe for measuring RGS domain-catalyzed GTP hydrolysis by Galphao. We demonstrate the effectiveness of this assay in the analysis of loss-of-function point mutants of both Galphao and RGS12. This assay should be useful in screening for and analyzing RGS protein inhibitory compounds.     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

A simple rapid fluorescent assay for adenosine deaminase activity.

A simple, rapid (2 hours), fluorescent test for the activity of blood adenosine deaminase (ADA) is described. The test which can be performed on both heparinized and dried blood, is based on the conversion of adenosine to inosine and ammonium in the presence of ADA. The enzyme activity is visually estimated by the oxidation of NADH (fluorescent) to NAD+ (non-fluorescent) in a coupled reaction with glutamate dehydrogenase. The disappearance of fluorescence indicates ADA activity in the sample. The advantages are discussed of the use of this test for the study of the autosomal recessive severe combined immunodeficiency.