Determination of serum aminotransferases: activation by pyridoxal-5'-phosphate in relation to substrate concentration.

1979 ◽  
Vol 25 (1) ◽  
pp. 55-59 ◽  
Author(s):  
J C Hafkenscheid ◽  
C C Dijt
Keyword(s):  
Enzymatic Activity ◽  
Serum Sample ◽  
Aspartate Aminotransferase ◽  
Substrate Concentration ◽  
Enzymatic Reaction ◽  
Reaction Medium ◽  
Aminotransferase Activity ◽  
Serum Aminotransferases ◽  
Stimulation Of

Abstract To investigate the activation of aspartate- and alanine aminotransferases by pyridoxal-5'-phosphate, we determined the enzymatic activity in serum in two different ways: (a) Preincubation of the serum alone or the serum with pyridoxal-5'-phosphate and starting the reaction by the addition of the serum sample or the serum sample + coenzyme, respectively. (b) Preincubation of the serum or the serum with pyridoxal-5'-phosphate in the reaction medium and starting the reaction by adding 2-oxoglutarate. There are only small differences in activities of both aminotransferases determined according to these two different methods. The stimulation by pyridoxal-5'-phosphate is also of the same order, when both methods are compared. Further, these enzymatic activities were measured with use of various concentrations of substrates. From our experiments we conclude that the degree of stimulation of the apoenzyme of the two enzymes is independent of which way the enzymatic reaction is carried out or the substrate concentration, except that aspartate aminotransferase activity is more stimulated by the coenzyme at higher 2-oxoglutarate concentrations.

COALESCENCE AS A FACTOR IN SOLVENT STIMULATION OF PLASTID PHOSPHATIDASE C ACTIVITY

1957 ◽  
Vol 35 (2) ◽  
pp. 119-126 ◽  
Author(s):  
Morris Kates ◽  
Paul R. Gorham
Keyword(s):  
Enzymatic Activity ◽  
Petroleum Ether ◽  
Ethyl Ether ◽  
Enzymatic Reaction ◽  
Ethyl Butyrate ◽  
Insoluble Complex ◽  
Ether Extraction ◽  
Propyl Ketone ◽  
Stimulation Of

Solvents which stimulate plastid phosphatidase C activity (e.g., ethyl ether, propyl ketone, and ethyl butyrate) have been observed (microscopically) to produce coalescence of lecithin and chloroplast phases, while those which do not stimulate the reaction (e.g., chloroform and petroleum ether) do not produce coalescence. In the ethyl ether-stimulated reaction, all of the original enzymatic activity was associated with the coalesced material, and the lecithin apparently formed an ether-insoluble complex with the chloroplasts; no enzymatic activity appeared in either the aqueous or ether phases. Ether extraction of chloroplasts alone did not liberate the enzyme from the plastids, and the presence of this stimulating solvent was still necessary for reaction with the substrate to occur. It is concluded that stimulating solvents achieve their effect primarily by causing substrate and plastid phases to coalesce and that the enzymatic reaction actually proceeds in the coalesced phase.

scholarly journals Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

2012 ◽  
Vol 32 (4) ◽  
pp. 661-667 ◽  
Author(s):  
Alexandre José Cichoski ◽  
Renata Bezerra Rotta ◽  
Gerson Scheuermann ◽  
Anildo Cunha Junior ◽  
Juliano Smanioto Barin
Keyword(s):  
Hydrogen Peroxide ◽  
Glutathione Peroxidase ◽  
Enzymatic Activity ◽  
Frozen Storage ◽  
Potassium Phosphate ◽  
Reaction Medium ◽  
Experimental Conditions ◽  
Future Studies ◽  
Different Sources

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC), the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Effects of Buffers on Aspartate Aminotransferase Activity and Association of the Enzyme with Pyridoxal Phosphate

1975 ◽  
Vol 21 (11) ◽  
pp. 1585-1591 ◽  
Author(s):  
Robert Rej ◽  
Raymond E Vanderlinde
Keyword(s):  
Schiff Base ◽  
Aspartate Aminotransferase ◽  
Phosphate Buffer ◽  
Pyridoxal Phosphate ◽  
Human Origin ◽  
Aminotransferase Activity ◽  
Factors Influencing ◽  
Absorbance Spectra ◽  
Stimulation Of

Abstract Using purified enzymes of human origin and patients’ sera, we examined factors influencing the in vitro association of pyridoxal phosphate with aspartate aminotransferase (EC 2.6.1.1). The rate of association was markedly retarded by phosphate buffer in comparison with tris(hydroxymethyl)aminomethane or six other buffers. Pyridoxal phosphate at an incubation concentration of 130 µmol/liter reactivated the entire apoenzyme portion of an apoenzyme/holoenzyme mixture within 5 min in tris(hydroxymethyl)aminomethane; in contrast, less than 20% was associated during 15 min in phosphate. Activity measured in tris(hydroxymethyl)aminomethane-buffer without exogenous pyridoxal phosphate was 4% greater than that in phosphate and was slightly increased by increasing the pH of the assay mixture from 7.5 to 8.0. Aspartate in the incubation medium did not retard the stimulation in tris(hydroxymethyl)aminomethane buffer. While the magnitude of stimulation varied greatly among sera, a consistent mean stimulation of 30% for groups of sera with normal activities was found when asparate at 125 mmol/liter, 2-oxoglutarate at 6.7 mmol/liter and tris(hydroxymethyl)aminomethane at 90 mmol/liter were used, an increase over the 16% with phosphate buffer [Clin. Chem. 19, 92 (1973)]. Absorbance spectra suggest pyridoxal phosphate exists as the Schiff base of tris(hydroxymethyl)aminomethane or aspartate, or both, under conditions of assay incubation (without addition of 2-oxoglutarate). Nonenzymatic catalysis of the reaction by pyridoxal phosphate alone or a formation of a protein/pyridoxal phosphate adduct was discounted with use of D-asparate substrates

COALESCENCE AS A FACTOR IN SOLVENT STIMULATION OF PLASTID PHOSPHATIDASE C ACTIVITY

1957 ◽  
Vol 35 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Morris Kates ◽  
Paul R. Gorham
Keyword(s):  
Enzymatic Activity ◽  
Petroleum Ether ◽  
Ethyl Ether ◽  
Enzymatic Reaction ◽  
Ethyl Butyrate ◽  
Insoluble Complex ◽  
Ether Extraction ◽  
Propyl Ketone ◽  
Stimulation Of

Solvents which stimulate plastid phosphatidase C activity (e.g., ethyl ether, propyl ketone, and ethyl butyrate) have been observed (microscopically) to produce coalescence of lecithin and chloroplast phases, while those which do not stimulate the reaction (e.g., chloroform and petroleum ether) do not produce coalescence. In the ethyl ether-stimulated reaction, all of the original enzymatic activity was associated with the coalesced material, and the lecithin apparently formed an ether-insoluble complex with the chloroplasts; no enzymatic activity appeared in either the aqueous or ether phases. Ether extraction of chloroplasts alone did not liberate the enzyme from the plastids, and the presence of this stimulating solvent was still necessary for reaction with the substrate to occur. It is concluded that stimulating solvents achieve their effect primarily by causing substrate and plastid phases to coalesce and that the enzymatic reaction actually proceeds in the coalesced phase.

Increased Aspartate Aminotransferase Activity of Serum after in Vitro Supplementation with Pyridoxal Phosphate

1973 ◽  
Vol 19 (1) ◽  
pp. 92-98 ◽  
Author(s):  
Robert Rej ◽  
Charles F Fasce ◽  
Raymond E Vanderlinde
Keyword(s):  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Colorimetric Method ◽  
Small Range ◽  
Aminotransferase Activity ◽  
Kinetic Assay ◽  
Serum Supplementation ◽  
Sample Dilution ◽  
Stimulation Of

Abstract We examined the effect of pyridoxal phosphate supplementation on the apparent aspartate aminotransferase (EC 2.6.1.1.) activity of human serum. Supplementation by 25 µmol/liter effected an average increase of 16% in the results for kinetic assay. The increase was not the result of increased enzymatic or nonenzymatic blanks, and, within a small range, sample dilution had no significant effect. Part of the increase was attributable to the enzyme being protected against the loss of activity that occurs during preincubation with L-aspartate. A similar increase was not demonstrated in a two-point colorimetric method, perhaps because of the short reaction time, without preincubation, and the initial presence of both substrates in the assay. We attempted to Correlate such stimulation of aminotransferase activity and the patient’s diagnosis or treatment. Pyridoxal phosphate should be included in the reaction mixture when aspartate aminotransferase activity is being measured clinically

scholarly journals PRODUKSI DAN PENENTUAN KONDISI OPTIMUM ENZIM XILANASE Bacillus amyloliquefaciens FUKUMOTO PADA SUBSTRAT XILAN JERAMI

2015 ◽  
Vol 2 (1) ◽  
pp. 74
Author(s):  
Widiyanti Sekatresna ◽  
Abdi Dharma ◽  
Periadnadi
Keyword(s):  
Enzyme Activity ◽  
Rice Straw ◽  
Incubation Time ◽  
Substrate Concentration ◽  
Bacillus Amyloliquefaciens ◽  
Specific Activity ◽  
Enzymatic Reaction ◽  
Optimal Conditions ◽  
Enzyme Specific Activity

 ABSTRACT The production and determination of  optimal condition of xylanase produced by Bacillus amyloliquefaciens on rice straw xylan were investigated in this study. The parameters to be observed were optimal conditions of pH, temperature, substrate concentration and incubation time. Xilanase activity was determined by measuring the amount of reducing sugar formed in the enzymatic reaction based on Somogyi Nelson method. Optimal conditions needed for the production of xylanase were at pH 7, temperature 27°C and six days of incubation time. While optimal conditions of xylanase action were reached at pH 8.2, temperature 45°C, substrate concentration 3.5%(w/w) and 15 minutes of incubation time with enzyme activity and enzyme specific activity of 1.285 U/mL and 0.738 U/mg respectively. As a comparison, xylanase was also produced on pure xylan  (birchwood), enzyme activity and enzyme specific activity obtained were 2.701 U/mL and 1.658 U/mg respectively. Cellulase content in enzyme produced on rice straw xilan showed the enzyme activity of 0.094 U/mL.  Keywords : xylanase, Bacillus amyloliquefaciens, rice straw xilan

A Method for the Colorimetric Determination of β-Glucuronidase in Urine, Serum, Tissue; Assay of Enzymatic Activity in Health and Disease

1959 ◽  
Vol 36 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Julius A. Goldbarg ◽  
Esteban P. Pineda ◽  
Benjamin M. Banks ◽  
Alexander M. Rutenburg
Keyword(s):  
Enzymatic Activity ◽  
Colorimetric Determination ◽  
Health And Disease ◽  
Urine Serum
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