Transaminases, activity determinations

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). 

In uremia there are present in the serum a variety of known and unknown metabolites that can produce aberrant laboratory results. Significant differences in glucose concentration have been observed in such specimens analyzed by ferricyanide (F2a) or Fe (II)-5-pyridylbenzo-diazepin-2-one reduction methods compared to glucose oxidase procedures (K7a). In a patient with elevated creatinine (15 mg/100 ml) and uric acid (10 mg/100 ml), the glucose value determined by the automated alkaline ferricyanide procedure was overestimated by 20 mg/100 ml (C4). In uremic patients undergoing chronic hemodialysis there is a decrease in transaminase activity. In 11 of 19 such patients, there was... 

Figure 24.10. Stereochemistry of Proton Addition. In a transaminase active site, the addition of a proton from the lysine residue to the bottom face of the quinonoid intermediate determines the 1 configuration of the amino acid product. The conserved arginine residue interacts with the a-carboxylate group and helps establish the appropriate geometry of the quinonoid intermediate.

Fig. 5. A Time course of the reduction in activity of acetylomithine S-transaminase by treatment of arginine-grown cells of the argR strain 961 of Escherichia cott with 0.4 M Mg + plus L-arginine hydrochloride (0.1 mg/ml), at 37° with aeration. The transaminase activity is determined [8] in extracts. B Restoration of acetylomithine 8-transaminase activity. An extract, prepared after treatment of cells wdth Mg + plus arginine for 2 hours, is kept at 0° and assayed at intervals. The activity of the extract is plotted as a percentage of that of an untreated-control extract (which remains approximately constant). From Leisinger et al. [67].

High-Throughput Assay to Determine Transaminase Activity... 

Speedie (4S) has obtained a cell-free preparation from S. flocculus which catalyzes the formation of (31) from L-tryptophan and S-adenosyl-L methionine. The crude enzyme has been purified 2-fold by ammonium sulfate fractionation, and preliminary results with this preparation after dialysis indicated that pyridoxal phosphate is not required, but may cause some stimulation of enzyme activity. At this stage of purification tryptophan transaminase activity was also present, and it has not yet been possible to determine whether the true methylase substrate is an activated tryptophan, or indole pyruvic acid (33), as has been demonstrated to be the case in the biosynthesis of indolmycin (34) (46). 

Activities of glutamate-pyruvate transaminase (SGPT, GPT) (EC 2.6.1.2), L-y -glutamyl-transferase (y-GT) (EC 2.3.2.2) and level of triglycerides (TG) in serum, as well as levels of glutathione (GSH) and malondialdehyde (MDA) in the liver were determined. 

Clinical Biochemical Determinations of the Serum Serum lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities were measured on fresh, refrigerated serum within U8 h of sacrificing the animal. Lactic dehydrogenase was measured according to the method of Amador, Dorfman, and Wacker (12). Serum GPT activity was measured according to the method of Wroblewski and LaDue (13). 

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... 

Throughout the text, serum aldolase has been determined by the spec-trophotometric method (BIO, L7, S29) and both transaminases by the colorimetric method (R2). The normal ranges (R13) in conventional units are for serum aldolase 2.3-8.8 units per ml (mean 5.7 units), for SCOT 12-36 units per ml (mean 19 units), and for SGPT 4-24 units per ml (mean 12 units). In each figure the activity of serum aldolase is denoted by an uninterrupted line, of SCOT by long strokes, and of SGPT by short strokes (Figs. 7-15). 

Amino acids constitute the second largest source of nonprotein nitrogen in serum, urea being major source. The determination of total serum amino acids can provide valuable clinical information. Single amino acids are measured to gain access to particular enzyme activities of transaminases and peptidases for instance. Amino acids are also important in the food industry and in biotechnology. Their concentration in food can be used as a measure of the nutritive value of the food. 

The course of these experiments can best be illustrated by some examples of the changes in the senim glutamic-oxalacetic transaminase (GOT) activity. GOT activity has proved to be a good indication of the tissue destruction which occurs. The normal values for serum GOT activity in pigs, as determined by the method of Ordell (1956) at SVA, is 22.5 7.7 units (n = Gl). 

The authors also measured the activity of serum glutamic oxalacetic transaminase (SCOT), creatine phosphokinase (CPK), and lactic dehydrogenase (LDH) to determine if there was evidence of myocardial necrosis. They... 

Determination of ALT ALT (formerly glutamate pyruvate transaminase) catalyzes the equilibrium transfer reaction of the amino group from L-alanine to 2-oxoglutarate to form L-glutamate and pyruvate ALT requires pyridoxal phosphate as coenzyme, which acts as an amino carrier. It is found in the main organs, such as the liver, kidney, and heart. The ALT activity in serum is elevated in diseases of the liver. 

A great deal of early research in the field established the foundations that now govern the concept of enzyme mimicry using MIPs, including the importance of structure-function relationships in determining binding and catalytic activity. Nicholls etal. prepared and evaluated an MIP transaminase mimic for the reaction of phenylpyruvic acid and pyridoxamine (Scheme 22). ... 

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