Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.4.1.2 (glutamate dehydrogenase)
 4,380 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a coupled-enzyme equilibrium method for measuring urea in serum, which is performed on supernates prepared by treating each specimen with Ba(OH)2 and ZnSO4 (Somogyi reagent). Analytical recovery of [14C]urea added to a variety of matrices was essentially complete (mean, 100.6%) for the supernates after precipitation. Nine variables were univariately examined in arriving at the reaction conditions for the method: glutamate dehydrogenase, urease, 2-oxoglutarate, ADP, Tris . HCI, NADH, EDTA, pH, and temperature. The reagent is stable for at least 48 days at--20 degrees C and for 23 days at 4 degrees C. Mean analytical recovery of urea (14 mmol/L) added to seven different specimens (three different matrices) was 100.8%. The analytical linear range of the method extends to 30 mmol of urea per liter. Of 22 potential interferents, only bilirubin at 1 mmol/L (580 mg/L), hemoglobin at 10 g/L, and hydroxyurea at 6 mmol/L showed more than 2% interference. We discuss precision and effects of specimen dilution, and compare results for 100 human serum specimens with those measured for the same specimens with four other urea methods. We examined the effects of measuring a blank, consisting of sample and reagent without urease, with each specimen.
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PMID:A coupled-enzyme equilibrium method for measuring urea in serum: optimization and evaluation of the AACC study group on urea candidate reference method. 737 2

A strategy for the multifunctionalization of the FIA biosensor was developed. The described multifunctional FIA system offers a fast and simple method for the simultaneous determination of ammonia, creatinine, and urea. The hydrolysis of creatinine by creatinine deiminase (CRDI) or of urea by urease forms ammonia, which is amperometrically detected by an oxygen electrode, based on an enzyme conversion system, glutamate dehydrogenase (GLDH)/glutamate oxidase (GLOD). The split of the stream into three after sample injection and confluence before the GLDH reactor resulted in a three-channel system, into which were set three parallel columns, respectively, filled with immobilized CRDI, urease, and CPG. A triple-peak recording was obtained by putting two delay coils at the channels involving CRDI and urease. Thus the interfering of the endogenous ammonia on the creatinine and urea assay is simultaneously compensated. Furthermore, the problem of great difference in concentration between urea and the other two components is resolved by taking advantage of the differentiated dilution effect for each channel caused from the split-stream, flow-injection system. Linear calibration ranges for ammonia, creatinine, and urea were 0.1-5, 0.2-10, and 2-40 mM, respectively. One run was finished within 5 minutes, and the system was reproducibility good (3 to 5%). The results of the urine assay obtained by the present method will be described in the near future.
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PMID:A multifunctional flow-injection biosensor for the simultaneous determination of ammonia, creatinine, and urea. 778 57

A two-step method for assaying creatinine in serum and urine samples, suitable with automated analyzers, is reported. Reagent 1, for the first step, contains a blanking system [creatine amidinohydrolase (CRTase), urease, glutamate dehydrogenase, NADPH, and 2-oxoglutarate] and a NADPH-regenerating system [Mg(2+)-dependent isocitrate dehydrogenase (ICD), MgCl2, and excess isocitrate]. Reagent 2, for the second step, contains the metal-chelating reagent trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CyDTA) and a trigger system [creatinine amidohydrolase (CRNase)]. When a specimen is mixed with reagent 1, all the creatine, urea, and NH3 present are removed by the blanking and NADPH systems. On adding reagent 2, CyDTA inactivates ICD to inhibit the NADPH system. Simultaneously, the creatinine (1 mol) in the specimen is hydrolyzed into creatine by CRNase, and then releases NADP+ (2 mol) through the blanking system. Our optimized method can determine creatinine linearly up to 500 mg/L, with within-day CVs < 1.2% and day-to-day CVs < 2.7%.
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PMID:Enzymatic rate assay of creatinine in serum and urine. 840 98

In Klebsiella aerogenes, the formation of a large number of enzymes responds to the quality and quantity of the nitrogen source provided in the growth medium, and this regulation requires the action of the nitrogen regulatory (NTR) system in every case known. Nitrogen regulation of several operons requires not only the NTR system, but also NAC, the product of the nac gene, raising the question of whether the role of NAC is to activate operons directly or by modifying the specificity of the NTR system. We isolated an insertion of the transposon Tn5tac1 which puts nac gene expression under the control of the IPTG-inducible tac promoter rather than the nitrogen-responsive nac promoter. When IPTG was present, cells carrying the tac-nac fusion activated NAC-dependent operons and repressed NAC-repressible operons independent of the nitrogen supply and even in the absence of an active NTR system. Thus, NAC is sufficient to regulate operons like hut (encoding histidase) and gdh (encoding glutamate dehydrogenase), confirming the model that the NTR system activates nac expression and NAC activates hut and represses gdh. Activation of urease formation occurred at a lower level of NAC than that required for glutamate dehydrogenase repression, and activation of histidase formation required still more NAC.
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PMID:The product of the Klebsiella aerogenes nac (nitrogen assimilation control) gene is sufficient for activation of the hut operons and repression of the gdh operon. 845 54

Amperometric enzyme probes for ammonium and urea have been assembled and evaluated using immobilized glutamate dehydrogenase and urease enzymes coupled with platinum electrodes. Analytical parameters such as pH, buffer, temperature, probe life-time, enzyme immobilization, cofactor concentration and response time have been optimized. Ammonium was detected in the range 10(-5)-3 x 10(-4) mol l-1. Better reproducibility and stability were achieved using the enzyme GLDH type III and NADH at a concentration of 10(-3) mol l-1. Urea has been determined in the range 10(-5)-3 x 10(-4) mol l(-1) using the enzyme urease first in solution and then immobilized on nylon net. The analysis was based on an amperometric measurement which gives a linear relationship between current and analyte concentration. This considerably improved the sensitivity of the analysis when compared with the potentiometric-based procedures. Moreover, this method does not suffer from the potassium ion interference which affects the potentiometric nonactin-based NH+4 electrodes. Analysis of ammonium and urea were carried out in standard solutions and in saliva samples. Results compared with a spectrophotometric reference procedure correlated well.
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PMID:Amperometric ammonium ion and urea determination with enzyme-based probes. 860 Sep 14

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


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