Effects of Pyridoxal Phosphate and L-Dopapyridoxal Phosphate on DOPA Decarboxylase Activity
NGO TRAN
Experientia 15.9. 1972 Specialia (28/9) 1021

The conversion of 3, 4-dihydroxyphenylalanine (DOPA) to dopamine by DOPA decarboxy- -lase is known in animal (1) and human (2 tissues. The maximum DOPA decarboxylase activity is obtained by the addition of pyridoxal phosphate (PLP) to the reaction mixture, but the degree to which the enzyme is activited depends on PH, substrate, source of enzyme, and other factors (3). There have been many reports concerning compounds which inhibit the enzymatic decarboxylation, particularly of DOPA (4). In the present study, we wish to report our observations on the continuous effect of PLP and L-DOPA-PLP solution on the decarbo -xylation of L-DOPA in the rat liver respectively.
Methods and materials. Details of the apparatus devised for an instantaneous and continuous measurement of 14CO2 production from DOPA-carboxyl-14C have been published previously (5, 6). For experimental procedure, rat liver homogenates were incubated with or without PLP, PLP plus L-DOPA, and L-DOPA in N2, at 37oC for 10 min. After 10 min of incubation, 0.12µCi DOPAcarboxyl-14C (specific activity: 3.4 µCi/mmole) was added to the incubation chamber with an enzyme concentration of 2.5 mg tissue/mL The total volume of incubation mixture was 40 ml. Continuous plotting of the 14CO2 data was achieved by a chart recorder.
Results. The Figure represents data showing changes in rates of 14CO2 production from DOPA-carboxyl-14C when this labeled substrate was incubated with rat liver homogenates with or without PLP, L-DOPA, and L-DOPA plus PLP.
As shown in the Table, there was no change in 14CO2 production (p > 0.05) when 0.6 x 10-6mole PLP was incubated with liver homogenates as compared to values obtained without PLP.
Similarly, an unchanged 14CO2 production (p > 0.05) was obtained in the presence of 15 X 10 -6 mole L-DOPA.
A decreased 14CO2 production was obtained in the presence of either 2.5 mM PLP (p < 0.001) or 0.6 mM PLP
plus 15 x 10 -6 mole L-DOPA (p < 0.001), respectively.
Cumulative 14CO2 production during initial 120 min from DOPA-carboxyl- 14C incubated with rat liver homogenates, with or without L-DOPA, pyridoxal phosphate, and L-DOPA plus pyridoxal phosphate, respectively. Each series of studies consist of 4 experiments. 14C production in 120 min is expressed as (percentage ± S.E.)/g tissue. Results of t-test for probability of significance are made between means of each experimental group compared to the control group.
Category: 14C production in 120 min
Control : 9.160 ± 0.398
0.6 mM PLP: 7.893 + 0.426 (p> 0.05)
0.6 mM PLP + 1.5 µmoles L-Dopa: 4.375 ± 0.187 (p <0.001}
15 µmoles L-DOPA: 8.863 ± 0.932 (p > 0.05)
2.5 mM PLP: 3.443 ± 0.142 (p < 0.001)
Time. 120m/n
Composite data of the rates of 14CO2 production from DOPA-earboxyl-t4C incubated with or without PLP, L-DOPA, and PLP plus L-DOPA in a 0.1M phosphate buffer, pH 7.0. The ordinate represents the percent of incubated I~C produced as ltCO2/min/g tissue, and the abscissa represents time in minutes after the administration of DOPA-carboxyl-14C. Each point represents the mean of the 14CO 2 production of 4 experiments at the given time and the length of the vertical bar through each point represents • 1 standard error of the mean
Discussion. It is known that L-DOPA was generally added to the enzyme sample incubated with 14C-labeled DOPA for the measurement of DOPA decarboxylase Activity(2).
L-DOPA is also known to inhibit the decarboxylation of DOPA both in vivo and in vitro studies (7). We have observed, however, that small doses of L-DOPA did not influence DOPA decarboxylase activity of rat liver homogenates in 0.1 M phosphate buffer at PH 7.0. But a marked inhibition of the enzyme was found after the aministration of 0.6mM of PLP plus the same amnount of L-DOPA. Such en enzyme inhibition caused by condensation products formed by L-Dopa and PLP in the incubation chamber, such as L-DOPA-PLP comlexe? It is well established that L-DOPA and m-hydroxyphenylethylamines react nonenzymatically with aldehydes including PLP to hydroxyisoquinoline derivatives (8). These products are stable (7.8), and can account for the inhibition of enzymatic decarboxylation obtained in our in-vitro studies. Such an inhibition of DOPA decarboxylase by L-DOPA plus PLP may not be caused by an enlarged pool of L-DOPA subsequent to the addition of L-DOPA mixture, since no change in the decarboxylation of DOPA was found with such a very small amount of L-DOPA (Figure).
The fact that DOPA decarboxylase activity is inhibited by L-DOPA plus PLP, possibly by condensation products formed from these substrates (7,8) may support previous clinical (9) and experimental (10) studies showing that pyridoxine antagonized the activity of L-DOPA in the treatment of parkinsonism. Interestingly the salutary effects and the serious side effects observed in patients treated in L-DOPA were also reserved by pyridoxine ( 10, 11).
References
1 M. SADDLER and C. R. J. RUTHVE, Progress in Medicinal Chemistry
(Ellis and West, London 1969), p. 200.
2 W. H. VOGEL, H. MACFARLAND and L. N. PRINCX, Biochem.
Pharmac. 19, 618 (1970).
3 j. AWAPARA, R. P. SANDMAN and C. HANLY, Archs Biochem.
Biophys. 98, 520 (1962).
4. W. G. CLARK and R. S. POGRUD, Circulation Res. 9, 721 (1961).
5 N. TRAN, Analytical Bioehem. 48, 112 (1972).
6 N. TRAN, J. Nuclear. Med. 13, 349 (1972).
v j. H. FELLMAN and E, S. ROTH, Biochemistry 10, 408 (1971).
Sommaire: Les résultants obtenus démontrent une inhibition de l’activité de la DOPA décarboxylase par de haute concentration de PDL or de faible concentration de L-DOPA plus PLD. Ceci pourrait expliquer les observations cliniques et experimentales précédentes demontrant que la pyridoxine antogonise l’effect de L-DOPA.utilisé dans le traitement de la maladie de Parkinson.
NGO TRAN
Département de Médecine Nucléaire et de Radiobiologie, Centre Hospitalier Universitaire de Sherbrooke (Québec, Canada) 10 February 1972 .12.4/448 - Release Date: 9/14/2006