Glutamic dehydrogenase assay

Very low concentrations of substrates may be assayed by recycling the test substrate for an appreciable but definite period of time and measuring the amount of product formed. The coenzyme NADPH, for instance, may be assayed using the two enzymes glutamate dehydrogenase (EC 1.4.1.3) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) ... 

E2. Ellis, G., and Goldberg, D. M., Optimal conditions for the kinetic assay of serum glutamate dehydrogenase activity at 37°C. Clin. Chem. 18, 523-527 (1972). 

Many enzymatic assays have also been developed for the analysis of proteolytic products. Total amino acids in Cheddar cheese were determined by Puchades et al. (1990) using the L-amino acid oxidase enzyme. Glutamic acid has been quantified by flow injection analysis using glutamate dehydrogenase (Puchades et al., 1989) and using the Boehringer-Mannheim kit (McSweeney et al., 1993). 

B.B. Rodriguez, J.A. Bolbot and I.E. Tothill, Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples, Biosens. Bioelectron., 19 (2004) 1157-1167. 

The catalytic detection of ammonium ions has not been extensively investigated in contrast with the large variety of potentiometric and amperometric chemical sensors and optical sensors described in the literature [236], Similarly, the detection of ammonia in air has merited diflierent approaches in the field of chemical sensors. Screen-printed electrodes modified with Meldola s Blue and covered with a polycarbonate membrane constitute the basis of the catalytic detection of NHj. The measurement is based on the electrocatalytic reduction of NADH upon addition of glutamate dehydrogenase to a stirred solution containing NADH, 2-oxoglutarate and ammonium ions. The rate of current decrease (nA s ), measured at 50 mV, correlates to the concentration of ammonium ions in the sample. Recoveries of ammonium ions in spiked pond and tap waters at the level of 0.1 ppm are close to 100%, which demonstrates the feasibility of this assay for the detection of ammonium ions in waters [237],... 

EN81 Bach, P.R. (1992). Interfering reactions in an ammonia assay using glutamate dehydrogenase. Clin. Chem. 38, 1024, Abstr. 383. 

Spectrophotometric approaches to ammonium quantitation include the Berthelot reaction and the enzymatic assay with glutamate dehydrogenase [L-glutamate NAD(P) oxidoreductase (deaminating), EC 1.4.1.3]. This latter approach has been accepted as a reference method and adapted to a range of analytical platforms. 

Tabata M, Kidneyo T, Totani M. Automated assay of creatinine in serum as simplified by the use of immobilised enzymes, creatinine deaminase and glutamate dehydrogenase. Anal Biochem 1983 134 44-9. 

Both enzymatic and chemical methods are used to measure ammonia in body Buids. Enzymatic assay with glutamate dehydrogenase is the most frequently used method. Plasma ammonia measurement is particularly susceptible to contamination, leading to falsely elevated concentrations. Some of the common samphng problems are discussed in the third edition of this textbook. 

This enzyme should not be confused with glutamate dehydrogenase E.C. 1.4.1.3 or glutamate dehydrogenase E.C. 1.4.1.4. which catalyse the same reaction utilising NADP instead of NAD. This enzyme may be assayed by u.v. spectroscopy [393]. 

Several enzymes are not detected when fresh mitochondria are assayed, but are detected when the preparations are aged, warmed, frozen, or otherwise maltreated. These treatments, that cause loss of some activities, permit measurement of latent enzymes, including ATPase, glutamic dehydrogenase, and DPN cytochrome reductase. Some of the latent enzymes are readily solubilized by disruption of the mitochondria, while others remain associated with large sedimentable fragments. ... 

Fig. 2. Reaction of 3 -p-fluorosulfonylbenzoyladenosine with bovine liver glutamate dehydrogenase. Glutamate dehydrogenase (021 mg/ml) was incubated with 3 -FSBA (0.496 mil/) at 24° in 0.01 M sodium barbital buffer (pH 8) containing 0.43 M KCl and 5% ethanol. At each indicated time, an aliquot was withdrawn, diluted 20-fold with Tris-0.1 M acetate buffer (pH 8) at 0°, and assayed (A) in the absence and (B) in the presence of 100 yM ADP. Inset Determination of the pseudo first-order rate constant from the decrease in activation by ADP. (Ft and Fo are the enzymic velocities measured in the presence of ADP and the given and zero time, respectively, and F > is the constant velocity at the end of the reaction. The pseudo first-order rate constant calculated is 0D351 min. ) Data are taken from P. K. Pal, W. J. Wechter, and R. F. Colman, Biochemistry 14, 707 (1975).

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