Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.3.11 (glutamate dehydrogenase)
 4,437 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We established a simple and rapid kinetic assay for measurement of calcium in serum by using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on inhibition of the enzyme by calcium. In the assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH; EC 1.4.1.4); then in the presence of urea amidolyase, urea, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion production was inversely proportional to calcium ion concentration in serum. The concentration of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then monitored the change of absorbance at 340 nm. The within-run CVs of this method were 1.7-3.2% (n = 10) at 1.53-3.08 mmol/L, respectively. Day-to-day (total) CVs were 2.8-4.1%. Analytical recovery was 92-112%. The presence of other ions, ascorbic acid, reduced glutathione, bilirubin, hemoglobin, citrate, lipemic material, or human serum albumin did not affect this assay system. The correlation between values obtained with our method (y) and o-cresolphthalein complexone method (CPC) (x) was: y = 1.001x + 0.077 mmol/L (r = 0.949, Sy[symbol: see text]x = 0.079, n = 100); with the other enzymatic method (x) it was: y = 0.952x + 0.021 mmol/L (r = 0.955, Sy[symbol: see text]x = 0.074, n = 100). The SEs for each method were: 0.025 mmol/L, our method; 0.023 mmol/L, CPC method; and 0.025 mmol/L, the other enzymatic method.
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PMID:New enzymatic assay for calcium in serum. 869 77

We developed a new simple assay for potassium ion in serum using urea amidolyase (UAL) from yeast sp. The method is based on activation of the enzyme by potassium ion. We eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH), and then monitored the production of ammonium ion by UAL, urea, ATP, bicarbonate and magnesium ions. Ammonium ion was produced proportional to the potassium ion concentration and was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH. We monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion to this assay was eliminated by adding glycoletherdiamine-N, N, N', N'-tetraacetic acid to the reaction. The within-assay coefficients of variation (CV) of this method were 0.9-1.55% (n = 10) at 3.32-6.18 mmol/L. Day-to-day CVs ranged from 1.49% to 2.46%. The analytical recovery was 96-108%. The correlation coefficient between the values obtained by our method (y) and those by the ion-selective electrode (ISE) method (x) was 0.994 (y = 1.032x-0.166 mmol/L, Syx = 0.110, n = 100). The presence of bilirubin, haemoglobin or other ions did not affect this assay, confirming the usefulness of this assay for clinical purposes.
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PMID:New enzymatic assay with urea amidolyase for determining potassium in serum. 924 70

Axenic mycelia of the ectomycorrhizal basidiomycete, Suillus bovinus, were grown in liquid media under continuous aeration with compressed air at 25 degrees C in darkness. Provided with glucose as the only carbohydrate source, they produced similar amounts of dry weight with ammonia, with nitrate or with alanine, 60-80% more with glutamate or glutamine, but about 35% less with urea as the respectively only exogenous nitrogen source. In crude extracts of cells from NH4(+)-cultures, NADH-dependent glutamate dehydrogenase exhibited high aminating (688 nmol x mg protein(-1) x min(-1)) and low deaminating (21 nmol x mg protein(-1) x min(-1)) activities. Its Km-values for 2-oxoglutarate and for glutamate were 1.43 mM and 23.99 mM, respectively. pH-optimum for amination was about 7.2, that for deamination about 9.3. Glutamine synthetase activity was comparatively low (59 nmol x mg protein(-1) x min(-1)). Its affinity for glutamate was poor (Km = 23.7 mM), while that for the NH4+ replacing NH2OH was high (Km = 0.19 mM). pH-optimum was found at 7.0. Glutamate synthase (= GOGAT) revealed similar low activity (62 nmol x mg protein(-1) x min(-1)), Km-values for glutamine and for 2-oxoglutarate of 2.82 mM and 0.28 mM, respectively, and pH-optimum around 8.0. Aspartate transaminase (= GOT) exhibited similar affinities for aspartate (Km = 2.55 mM) and for glutamate (Km = 3.13 mM), but clearly different Km-values for 2-oxoglutarate (1.46 mM) and for oxaloacetate (0.13 mM). Activity at optimum pH of about 8.0 was 506 nmol x mg protein(-1) x min(-1) for aspartate conversion, but only 39 nmol x mg protein(-1) x min(-1) at optimum pH of about 7.0 for glutamate conversion. Activity (599 nmol x mg protein(-1) x min(-1)), substrate affinities (Km for alanine = 6.30 mM, for 2-oxoglutarate = 0.45 mM) and pH-optimum (6.5-7.5) proved alanine transaminase (= GPT) also important in distribution of intracellular nitrogen. There was comparatively low activity of the obviously constitutive enzyme, urease, (42 nmol x mg protein(-1) x min(-1)) whose substrate affinity was rather high (Km = 0.56 mM). Nitrate reductase proved substrate induced; activity could only be measured after exposure of the mycelia to exogenous nitrate. Routes of entry of exogenous nitrogen and tentative significance of the various enzymes in cell metabolism are discussed.
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PMID:Investigations into enzymes of nitrogen metabolism of the ectomycorrhizal basidiomycete, Suillus bovinus. 1081 9

An enzymatic end-point method for the quantitative determination of L-arginine was evaluated with samples of synthetic wine and natural grape juice. The enzymes arginase, urease, and glutamate dehydrogenase were used in this simple assay, similar to those described for many metabolites by Boehringer-Mannheim. In synthetic wine, recovery of L-arginine ranged between 98.3 and 104.4% and the precision as coefficient of variation was between 0.4 and 1.47% in the concentration range of the method, 0-100 mg/L L-arginine. The recovery of L-arginine in a grape juice with added L-arginine after clarification with polyvinylpolypyrrolidone ranged between 100 and 101.3%, and the coefficient of variation was 0.6%. The method has low material costs of approximately 0.43 U.S.$ per assay, and the time course of the reaction facilitates measurement of several samples concurrently. The results of this evaluation indicate that the enzymatic assay is a preferred method over colorimetric methods for the manual determination of L-arginine.
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PMID:Quantitative determination of L-arginine by enzymatic end-point analysis. 1126 90

Immobilization of urease and glutamate dehydrogenase enzymes in electrochemically prepared polypyrrole-polyvinyl sulfonate films (PPY-PVS) was carried out using physical adsorption and electrochemical entrapment techniques. Detailed studies on optimum pH, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy of the enzymes in the immobilized state were conducted. The value of the apparent Michaelis-Menten constant was experimentally determined to be 2.5 and 2.7 for physically adsorbed and electrochemically entrapped urease in PPY-PVS films, respectively.
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PMID:Coimmobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrole-polyvinyl sulfonate films. 1178 91

We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP(+) in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N', N'-tetraacetic acid to the reaction system. The with-in-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively. The day-to-day CVs were 2.23-4.59%. Analytical recovery was 92-115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system. The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 +/- 0.86 mmol/L [(values by reference method - that of present method) +/- SD] using the Bland-Altman technique. J. Clin. Lab. Anal. 17:52-56, 2003.
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PMID:New enzymatic assay for serum urea nitrogen using urea amidolyase. 1264 Jun 27

In vitro toxic effects of sulfonylurea herbicides (thifensulfuron-methyl and metsulfuron-methyl) were evaluated according to a new protocol. Physiological conditions were reproduced in order to boost toxicovigilance. Sulfonylureas and their hydrolysis products were added to biological substrates such as urea, alanine, aspartic acid, alpha-ketoglutarate, oxaloacetate, pyruvate and then incubated with some specific enzymes. Addition of these sulfonylureas and their degradation products did not significantly change the enzymatic activity of the urease, aspartate-aminotransferase, glutamate dehydrogenase, malate dehydrogenase and lactate dehydrogenase. However, the acid hydrolysis products inhibited up to 95% of the activity of the alanine-aminotransferase at low concentrations (0.27 micromol L(-1)). Inhibition did not affect the mitochondrial aspartate-aminotransferase.
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PMID:Toxicovigilance: new biochemical tool used in sulfonylurea herbicides toxicology studies. 1476 45

An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH(4)(+) is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease activity. The presence of heavy metals in the samples inhibits the urease activity, resulting in a lower NH(4)(+) production and therefore a decrease in NADH oxidation. The use of metallised carbon electrodes gave a decrease in NADH oxidation potential from +300 mV versus Ag/AgCl compared with > +600 mV for bare carbon electrodes, and thus minimised interferences from oxidizable species present in the samples. Electrodes fouling and possible contamination after reuse and cleaning was also eliminated by using screen-printed disposable electrodes. The linear range obtained for Hg(II) and Cu(II) was 10-100 microgl(-1) with a detection limit of 7.2 microgl(-1) and 8.5 microgl(-1), respectively. Cd(II) and Zn(II) produced enzyme inhibition in the range 1-30 mgl(-1), with limits of detection of 0.3 mgl(-1) for Cd(II) and 0.2 mgl(-1) for Zn(II). Pb(II) did not inactivate the urease enzyme significantly at the studied range (up to 50 mgl(-1)). Coefficients of variation (CV) values were 6-9% in all cases. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach provides to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection.
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PMID:Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples. 1504 46

A novel assay method was investigated for urease (EC 3.5.1.5) from Pseudomonas aeruginosa and Canavalia ensiformis by Fourier transform infrared spectroscopy. This enzyme catalyzed the hydrolysis of urea in phosphate buffer in deuterium oxide ((2)H(2)O). The intensities of the bicarbonate bands maxima at 1625 and 1365 cm(-1) and of the amide I band at 1605 cm(-1) were measured as a function of time to study the kinetics of urea hydrolysis. The extinction coefficients epsilon of urea and bicarbonate were determined to be 0.72, 0.48, and 0.56 mM(-1)cm(-1) at 1625, 1605, and 1365 cm(-1), respectively. The initial velocity is proportional to the enzyme concentration by using the ureases from both C.ensiformis and P. aeruginosa. The kinetic constants (V(max), K(m), and K(cat)) determined by Lineweaver-Burk plot were 532.2 U mg(-1) protein, 6.4mM, and 806.36 s(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on glutamate dehydrogenase in aqueous media. Therefore, this spectroscopic method is highly suited to assay for urease activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of urease activity.
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PMID:The use of Fourier transform infrared spectroscopy to assay for urease from Pseudomonas aeruginosa and Canavalia ensiformis. 1524 3

A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)-glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L(-1) for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 microg L(-1) for Hg(II) and Cu(II), respectively, and 4.3 mg L(-1) for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (microg L(-1)) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted samples.
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PMID:Urease-glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples. 1530 Mar 52


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