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Effect of pyridoxal 5′-phosphate on the temperature relationships of alanine aminotransferase
329
Clinica
Chimica
0 Elsevler
Acta,
Scientific
64 (1975) 329-331 Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION
CCA 7314
EFFECT OF PYRIDOXAL 5’-PHOSPHATE ON THE TEMPERATURE RELATIONSHIPS OF ALANINE AMINOTRANSFERASE
K. JUNG and E. EGGER Department of Clinical Chemistry, Schumannstrasse 20/21, Postfach
(Received
Charitk, Humboldt 140 (Germany)
University
Berlin,
104 Berlin,
May 20, 1975)
The problem of temperature in the determination of enzyme activities is an active field of research in clinical chemistry today. The effect of temperature on the activity of serum enzymes has been, up to now, demonstrated for various enzymes [l--3]. In the choice of the temperature for the determination of enzyme activity, it is very important to take into account the complex relationship between temperature and the other conditions for determination of enzyme activities [3]. Our experiments [4] have shown that the influence of pyridoxal 5’-phosphate must also be taken into consideration for determination of aspartate arninotransferase (EC 2.6.1.1) activity. The present communication is concerned with the study of the effect of pyridoxal 5’-phosphate on the temperature behaviour of alanine aminotransferase (EC 2.6.1.2). Enzyme reaction rates were measured at 340 nm on the LKB reaction rate analyzer 8600 at 37°C and on the Eppendorf photometer at 334 nm (with recorder) at 25°C and at the temperatures indicated in the figure. The temperature in the cuvette was maintained constant (+O.O7”C) as previously described [3]. Unless otherwise stated, the final reaction mixture contained, per liter: 800 mmol of L-alanine; 80 mmol of phosphate buffer, pH 7.4; 0.18 mmol NADH; 12 mmol of 2-oxoglutarate; 2000 Ulactate dehydrogenase (EC 1.1.1.37) and 200 pmol pyridoxal 5’-phosphate. Ratio of sample volume to final volume was 1 : 7.5. After preincubation for 20-25 minutes, the reaction was started by addition of 2-oxoglutarate. The rate of decrease in absorbance was monitored continously. The enzyme activities were calculated from the reaction rates in the first minute. The nonspecific reaction rate and the apo-aminotransferase activity of lactate dehydrogenase were subtracted. For the Arrhenius plot, duplicate assays were performed. The standard deviations were calculated using the following formula s = d(R2/2m) (R, difference between duplicate assays; 112,number of duplicate assays). The serum samples were taken from patients. All reagents were obtained from C.F. Boehringer, Mannheim.
330
Pyridoxal 5’-phosphate is the coenzyme of aspartate aminotransferase and alanine aminotransferase [ 51. Addition of pyridoxal 5’-phosphate to the reaction mixture increases the activity of aspartate aminotransferase [6,7]. A recent study has also shown a stimulation of alanine aminotransferase activity when pyridoxal 5’-phosphate was added to the reaction mixture [S]. The variation of this stimulation with temperature has not hitherto been investigated. Sera from 31 different patients were assayed at 25°C and 37°C with or without the addition of pyridoxal 5’-phosphate. 13 samples showed activities above the upper limit of the normal range. Adding pyridoxal 5’-phosphate the mean stimulation of alanine aminotransferase activity was 3.5% i 1.5 at 25°C and 14.4% + 2.3 at 37°C (arithmetic mean * standard deviation of the mean). Use of the paired t-test showed the differences between the stimulation rates at 25°C and 37°C to be statistically significant (p < 0.005; t = 4.93). This difference of stimulation rates is not caused by a protective action of pyridoxal 5’-phosphate at higher incubation temperatures. We could find no decrease of alanine aminotransferase activity when sera were incubated at 37°C for 120 minutes. To find out the true stimulation of alanine aminotransferase activity effected by pyridoxal 5’-phosphate it was necessary to examine the relationship between temperature and alanine aminotransferase activity. We measured the alanine aminotransferase activity. We measured the alanine aminotransferase activity of pooled serum in the temperature range of 17”C-41°C The results are shown in Fig. 1 as an Arrhenius plot. By shifting the temperature from 17°C to 41°C for the determination of alanine aminotransferase activity, the ratio increases between measured activity with pyridoxal 5’-phosphate and the
Fig. 1. Arrhenius Plot for alanine aminotransferase activity with and without stimulation by pyridoxal B’-phosphate. Conditions are given in the text. Assays of enzyme activity were performed on an Eppendorf photometer with a thermostatted cell howing supplied from an external water bath. Standard deviation, 11.2 UP for activity measurements without pyridoxal 5’-phosphate and t1.8 U/l for measurements with pyridoxat 5’-phosphate.. u. without pyridoxd 5-phosphate: 0 ------0, with pyridoxal 5’-phosphate.
331
activity without pyridoxal5’-phosphate. The addition of pyridoxal 5’-phosphate prevents the deviation from the straight Arrhenius-plot at higher temperature. In the experiments without addition of pyridoxal 5’-phosphate this deviation lies between 27°C and 30°C [ 1,9]. Recently, Szasz [l] attempted to explain the causes of deviation in the Arrhenius plot at higher temperatures. He assumed that these may be due to alterations in protein structure and the optimal conditions for determination of enzyme activity. However, the effects of temperature are extremely complex [lo]. Our experiments [9] also suggest that the deviation of alanine aminotransferase activity from the straight line in the Arrhenius plot cannot be explained by alteration of substrate saturation with temperature. For alanine aminotransferase, the substrate concentration for L-alanine, 2-oxoglutarate and NADH was optimal [9]. This deviation, like that for aspartate aminotransferase .[4] disappears on adding pyridoxal 5’phosphate in vitro. With respect to the optimal conditions for different temperatures we must take into consideration the decrease at higher incubation temperatures in the affinity of the alanine aminotransferase for the coenzyme. Consequently the concentration and affinity of coenzyme also play an important role in the choice of temperature for the determination of alanine aminotransferase activity. References 1 2 3 4 5 6 7 8 9 10
G. Szasz, Z. Klin. Chem. Klin. Biochem., 12 (1974) 166 M.J. McQueen. Z. Klin. Chem. Klin. Biochem., 13 (1975) 17 K. Jung, E. Egger, R. Neumann and B. Liidtke, Z. Klin. Chem. Klin. Biochem.. 12 (1974) K. Jung. B. Liidtke and E. Egger, Z. Klin. Chem. Klin. Biochem., 13 (1975) in press D.E. O’Kane and I.C. Gunsahns, J. Biol. Chem., 170 (1947) 425 G. Laudahn and E. Hartmann, Klin. Wochenschr.. 48 (1970) 1010 R. Rej, C.F. Fasce and R.E. Vanderlinde, Clin. Chem., 19 (1973) 92 T. Cheung and M.H. Briggs, Clin. Chim. Acta. 54 (1974) 127 B. Liidtke, K. Jung and E. Egger, Rev. Room. Biochim.. in press M. Dixon and E.C. Webb, Enzymes. Longman, London, 1964, p. 158
159
Clinica
Chimica
0 Elsevler
Acta,
Scientific
64 (1975) 329-331 Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION
CCA 7314
EFFECT OF PYRIDOXAL 5’-PHOSPHATE ON THE TEMPERATURE RELATIONSHIPS OF ALANINE AMINOTRANSFERASE
K. JUNG and E. EGGER Department of Clinical Chemistry, Schumannstrasse 20/21, Postfach
(Received
Charitk, Humboldt 140 (Germany)
University
Berlin,
104 Berlin,
May 20, 1975)
The problem of temperature in the determination of enzyme activities is an active field of research in clinical chemistry today. The effect of temperature on the activity of serum enzymes has been, up to now, demonstrated for various enzymes [l--3]. In the choice of the temperature for the determination of enzyme activity, it is very important to take into account the complex relationship between temperature and the other conditions for determination of enzyme activities [3]. Our experiments [4] have shown that the influence of pyridoxal 5’-phosphate must also be taken into consideration for determination of aspartate arninotransferase (EC 2.6.1.1) activity. The present communication is concerned with the study of the effect of pyridoxal 5’-phosphate on the temperature behaviour of alanine aminotransferase (EC 2.6.1.2). Enzyme reaction rates were measured at 340 nm on the LKB reaction rate analyzer 8600 at 37°C and on the Eppendorf photometer at 334 nm (with recorder) at 25°C and at the temperatures indicated in the figure. The temperature in the cuvette was maintained constant (+O.O7”C) as previously described [3]. Unless otherwise stated, the final reaction mixture contained, per liter: 800 mmol of L-alanine; 80 mmol of phosphate buffer, pH 7.4; 0.18 mmol NADH; 12 mmol of 2-oxoglutarate; 2000 Ulactate dehydrogenase (EC 1.1.1.37) and 200 pmol pyridoxal 5’-phosphate. Ratio of sample volume to final volume was 1 : 7.5. After preincubation for 20-25 minutes, the reaction was started by addition of 2-oxoglutarate. The rate of decrease in absorbance was monitored continously. The enzyme activities were calculated from the reaction rates in the first minute. The nonspecific reaction rate and the apo-aminotransferase activity of lactate dehydrogenase were subtracted. For the Arrhenius plot, duplicate assays were performed. The standard deviations were calculated using the following formula s = d(R2/2m) (R, difference between duplicate assays; 112,number of duplicate assays). The serum samples were taken from patients. All reagents were obtained from C.F. Boehringer, Mannheim.
330
Pyridoxal 5’-phosphate is the coenzyme of aspartate aminotransferase and alanine aminotransferase [ 51. Addition of pyridoxal 5’-phosphate to the reaction mixture increases the activity of aspartate aminotransferase [6,7]. A recent study has also shown a stimulation of alanine aminotransferase activity when pyridoxal 5’-phosphate was added to the reaction mixture [S]. The variation of this stimulation with temperature has not hitherto been investigated. Sera from 31 different patients were assayed at 25°C and 37°C with or without the addition of pyridoxal 5’-phosphate. 13 samples showed activities above the upper limit of the normal range. Adding pyridoxal 5’-phosphate the mean stimulation of alanine aminotransferase activity was 3.5% i 1.5 at 25°C and 14.4% + 2.3 at 37°C (arithmetic mean * standard deviation of the mean). Use of the paired t-test showed the differences between the stimulation rates at 25°C and 37°C to be statistically significant (p < 0.005; t = 4.93). This difference of stimulation rates is not caused by a protective action of pyridoxal 5’-phosphate at higher incubation temperatures. We could find no decrease of alanine aminotransferase activity when sera were incubated at 37°C for 120 minutes. To find out the true stimulation of alanine aminotransferase activity effected by pyridoxal 5’-phosphate it was necessary to examine the relationship between temperature and alanine aminotransferase activity. We measured the alanine aminotransferase activity. We measured the alanine aminotransferase activity of pooled serum in the temperature range of 17”C-41°C The results are shown in Fig. 1 as an Arrhenius plot. By shifting the temperature from 17°C to 41°C for the determination of alanine aminotransferase activity, the ratio increases between measured activity with pyridoxal 5’-phosphate and the
Fig. 1. Arrhenius Plot for alanine aminotransferase activity with and without stimulation by pyridoxal B’-phosphate. Conditions are given in the text. Assays of enzyme activity were performed on an Eppendorf photometer with a thermostatted cell howing supplied from an external water bath. Standard deviation, 11.2 UP for activity measurements without pyridoxal 5’-phosphate and t1.8 U/l for measurements with pyridoxat 5’-phosphate.. u. without pyridoxd 5-phosphate: 0 ------0, with pyridoxal 5’-phosphate.
331
activity without pyridoxal5’-phosphate. The addition of pyridoxal 5’-phosphate prevents the deviation from the straight Arrhenius-plot at higher temperature. In the experiments without addition of pyridoxal 5’-phosphate this deviation lies between 27°C and 30°C [ 1,9]. Recently, Szasz [l] attempted to explain the causes of deviation in the Arrhenius plot at higher temperatures. He assumed that these may be due to alterations in protein structure and the optimal conditions for determination of enzyme activity. However, the effects of temperature are extremely complex [lo]. Our experiments [9] also suggest that the deviation of alanine aminotransferase activity from the straight line in the Arrhenius plot cannot be explained by alteration of substrate saturation with temperature. For alanine aminotransferase, the substrate concentration for L-alanine, 2-oxoglutarate and NADH was optimal [9]. This deviation, like that for aspartate aminotransferase .[4] disappears on adding pyridoxal 5’phosphate in vitro. With respect to the optimal conditions for different temperatures we must take into consideration the decrease at higher incubation temperatures in the affinity of the alanine aminotransferase for the coenzyme. Consequently the concentration and affinity of coenzyme also play an important role in the choice of temperature for the determination of alanine aminotransferase activity. References 1 2 3 4 5 6 7 8 9 10
G. Szasz, Z. Klin. Chem. Klin. Biochem., 12 (1974) 166 M.J. McQueen. Z. Klin. Chem. Klin. Biochem., 13 (1975) 17 K. Jung, E. Egger, R. Neumann and B. Liidtke, Z. Klin. Chem. Klin. Biochem.. 12 (1974) K. Jung. B. Liidtke and E. Egger, Z. Klin. Chem. Klin. Biochem., 13 (1975) in press D.E. O’Kane and I.C. Gunsahns, J. Biol. Chem., 170 (1947) 425 G. Laudahn and E. Hartmann, Klin. Wochenschr.. 48 (1970) 1010 R. Rej, C.F. Fasce and R.E. Vanderlinde, Clin. Chem., 19 (1973) 92 T. Cheung and M.H. Briggs, Clin. Chim. Acta. 54 (1974) 127 B. Liidtke, K. Jung and E. Egger, Rev. Room. Biochim.. in press M. Dixon and E.C. Webb, Enzymes. Longman, London, 1964, p. 158
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