Transaminase assay

When the substrate is availabT in either the d- or 1-racemic form, it is preferable to use the appropriate isomer rather than its mixture In a case of transaminase assays for GOT and GPT activity, for example, the initial assays used the d-1 amino acid as substrate, and a marked improvement in activity and linearity was found by Henry and co-workers when they used 1-aspartate or 1-alanine, respectively (28) ... 

Amador, E. and Wacker, W E. C. Serum glutamic-oxaloacetic transaminase activity. A new modification and an analytical assessment of current assay technics. Clin. Chem. (1962), 8, 343-350. 

Russell, C. D. Cotlove, E. Serum glutamic-oxaloacetic transaminase Evaluation of a coupled-reaction enzyme assay by means of kinetic theory. Clin. Chem. (1971), 17, 1114-1122. 

Addition of ethyl acetate to a specimen having a transaminase activity of 47 units was responsible for the following increases in enzyme activity 10 mg/100 ml, 60 units 20 mg/100 ml, 77 units 40 mg/100 ml, 107 units and 80 mg/100 ml, 150 units. Transaminase activity in these specimens determined by another method ranged from 32 to 34 units (C7). Thus, when serum from patients with ketosis is assayed for aspartate aminotransferase activity by the diazo method, false elevations of activity may be recorded due to reaction of acetoacetic acid. In Table 11 are shown some values obtained by the diazo method and by an ultraviolet NADH NAD aspartate aminotransferase technique (B12). Examination of the medical records of these patients indicated that they were either diabetics who were in ketosis or individuals who were eating very poorly and had some degree of starvation ketosis. Similar elevations have been observed in patients receiving p-aminosalicylic acid (G6). 

Schor DS, Struys EA, Hogema BM, Gibson KM, Jakobs C (2001) Development of a stable-isotope dilution assay for gamma-aminobutyric acid (GABA) transaminase in isolated leukocytes and evidence that GABA and beta-alanine transaminases are identical. Clin Chem 47 525-531... 

Comparable reports have prompted a questionnaire investigation of 770 patients with type 2 diabetes at the start of acarbose therapy (62). Patients with one or more susceptibility factors for liver damage underwent ultrasonography and autoantibody assays. There was silent liver disease in 13% and 20 patients had a fatty liver without hepatic disease. In 15% of these patients there were slight reversible changes in transaminase activity after acarbose. 

Reliable enzymatic assays for SeMet are not available as specific SeMet metabolizing enzymes have not been identified and enzymes such as glutamine transaminase react with Met equally as well as with SeMet (Blazon et al., 1994). However, with some enzymes reaction rates for SeMet and Met differ sufficiently to be of some use in SeMet analysis. For example, SeMet is a better substrate than Met for the a,y-elimination by i.-methionine y-lyase of Pseudomonas putida (Esaki et al., 1979). The adenosyl methionine transferase from rat liver reacts with L-SeMet at 51% of the rate with L-Met, and with the corresponding D-isomers at only 13 and 10% of the rate of L-Met (Pan and Tarver, 1967). Other adenosyl methionine transferases, such as that from yeast, react with SeMet more rapidly and with higher stereoselectivity than with Met, providing an indirect means for SeMet determination (Mudd and Cantoni, 1957 Sliwkowski, 1984 Uzar and Michaelis, 1994). 

In this experiment, you will perform a three-step purification of glutamate-oxaloacetate transaminase from pig heart. With each step in the purification, you will assay different fractions for GOT activity and total protein concentration. These values will then be used to calculate the total activity, specific activity, and percent yield of GOT following each purification step. The goal of the experiment is to achieve 80- to 100-fold purification of the enzyme with >5% yield. The overall effectiveness of the purification procedure will be analyzed by SDS-PAGE on the last day of the experiment. Be sure that you determine three different... 

Sizer, I. W., and Jenkins, W. T. (1962). Glutamic Aspartic Transaminase from Pig Ventricles Preparation and Assay of Enzymes. Methods Enzymol 5 677. Stryer, L. (1995). Amino Acid Degradation and the Urea Cycle. In Biochemistry, 4th ed. New York Freeman. 

In the widely used colorimetric assay of SGOT and SGPT (R2), serum is incubated at 37 °G with phosphate-buffered L-aspartate/a-oxoglutarate or DL-alanine/a-oxoglutarate, respectively, the reaction terminated after a definite interval by addition of 2,4-dinitrophenylhydrazine in dilute HCl, and after a period at room temperature the hydrazones of oxalace-tate or pyruvate so formed are treated with dilute alkali and the optical density measured against water at 505 mp the results are read from standard curves of optical density against transaminase activity. 

K4. Karmen, A., A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. ]. Clin. Invest. 34, 131 (1955). 

The most common assay systems consist of cell cultures that are treated with the drug in the culture medium. Toxicity is commonly assessed by release of intracellular enzyme, for example, lactate dehydrogenase or aspartate transaminase, into the culture medium or by other indicators such as decreases in the rate of radiolabeled amino acid or nucteotide precursor incorporation into macromolecules. A decrease in the intracellular uptake of the vital dye, neutral red, has also... 

Glutamic-oxalacetate transaminase (GOT) is released into the blood stream as a result of myocardial infarction. The enzyme is assayed in serum by following the decrease in the absorbance of NADH in the malic dehydrogenase (MDH)-coupled reaction sequence shown below. 

A 61-year-old man developed weakness, severe jaundice, pruritus, and weight loss over 2 weeks. He had started to take metoprolol, fosinopril, and diazepam for hjrpertension 5 weeks before. He had raised hepatic transaminases and bihrubin. A hver biopsy showed cholestasis in a normal cellular architecture. A lympho-cjTe transformation assay showed reactivity to fosinopril but not diazepam or metoprolol. Bihrubin concentrations took 4 months to normalize and pruritus persisted for 6 months. 

The electrochemical detection utilized the re-oxidation of hexacyano-ferrate(II) on a platinum electrode. For pyruvate determination this assay was extended to a 3-enzyme system by the addition of glutamate p5u-uvate transaminase, which produces alanine from pyruvate. All enz5unes were used in solution in a reaction chamber of approximately 2 pi directly in front of the electrode. The cofactor NAD" " was coupled to dextran with a molecular weight of 40,000 to avoid its replacement for each assay. As the sensor responded to L-alanine and pyruvate again a differential measurement was required when a sample contained both compounds. It was applied to off-line monitoring of a cultivation of S. cerevisiae and data showed good correlation to the photometric assays. 

NSCLC patient with a 40% reduction in undefined measurable disease. A weekly schedule has also been evaluated and an MTD of 245 mg/m2 defined (167). Thirty patients were treated for a median of 6 weeks, and grade 3 DLTs included vomiting, diarrhea, dehydration, and transient elevations in creatinine and transaminases. As with the previous schedule, 80% FT inhibition in PBMCs was noted at the higher dose levels (168). In addition, FT inhibition by a mean of 80% was observed in 14 posttreatment tumor samples at 2 h and persisted at 30% inhibition at 24 h. In some tumor samples, assessment of apoptosis by a DNA break labeling assay revealed induction of apoptosis after dmg exposure, for example, a refractory breast cancer patient with a 5-month minor response. Two other schedules (169, 170), weekly for 4 weeks followed by a 2-week rest and oral twice a day for 14 days every 21 days, have been evaluated with no objective responses reported. 

The assay system for measuring transaminase activity contains two amino acids and two oxo-acids. As there is no convenient method for assaying amino acids, formation or consumption of the oxo-acids is measured. Historically,... 

A Saturation of Biochemical Function. A reliable biochemical indicator is required. For niacin, which NAD" - or NADP -containing enzyme should be selected Which transaminase will be the indicator for pyridox-ine Which function of vitamin A should be j selected for retinol, vision in the rods or cell differentiation As noted from Table 8.2, many of the assays for vitamin status have significant limitations to estimate reliable doses. 

Employment of glutamate oxidase enables the sequential assay of both transaminases without the need to coimmobilize a second enzyme. Using a glutamate oxidase sensor, Yamauchi et al. (1984) found a preincubation of the sample of 30 min to be necessary. The preincubation time could be lowered to 10 min by using an optimized sensor configuration (Wollenberger et al., 1989). 

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