Brain Research, 107 (1976) 591-601
591
© ElsevierScientificPublishing Company,Amsterdam- Printed in The Netherlands
DECARBOXYLATION OF NEWLY FORMED DOPA BY CAUDATE NUCLEUS SYNAPTOSOMAL PARTICLES
SAKTI P. BAGCHI* ANDTHOMAS M. SMITH Research Division, Marcy Psychiatric Center, Marcy, N.Y. 13403 (U.S.A.)
(Accepted October 4th, 1975)
SUMMARY
The present study compares dopamine formation from two different sources of DOPA: preformed and added to the medium and that newly formed by the hydroxylations of phenylalanine or tyrosine. Synaptosomal preparations from rat brain caudate nucleus were incubated with all-labeled DOPA mixed as cosubstrate with either p4C]phenylalanine or [14C]tyrosine. Following the incubation, DOPA and dopamine were separated and their isotope ratios (isotope in phenylalanine (tyrosine) × 100/isotope in DOPA cosubstrate) were determined and compared. The results show that the ratio in dopamine was not only different from that in DOPA but was in fact 8.3-15.0 times higher. Although the absolute values of the isotope ratios were affected by any changes of the cosubstrate concentrations, the ratio in dopamine was found to be higher than that in DOPA at all the substrate concentrations tested. When the synaptosomes were separated post-incubation from the medium and analyzed, the ratio in dopamine was also found to be higher than that in DOPA. Also, the large difference between the intrasynaptosomal ratios persisted throughout the incubation period ranging from 10 to 45 min. The results suggest that the DOPA which is newly formed by the hydroxylations, (a) may not be freely exchangeable with added DOPA because of compartmentation and (b) may be preferentially decarboxylated to dopamine.
INTRODUCTION During the course of our investigation of phenylalanine hydroxylation by brain tissue, we incubated caudate nucleus homogenate with [14C]phenylalanine mixed * To whom correspondence should be addressed at: Rockland Research Institute, Building 37, Orangeburg, New York, N.Y. 10962, U. S.A.
592 with [3H]tyrosine as the cosubstrate. The results 2 indicated that the isotope ratio (laC/3H / 100) in the dopamine formed was higher than that in the tyrosine isolated. In an attempt to clarify the reason for the higher 14C content of dopamine we further investigated the double labeling of dopamine and tyrosine by caudate synaptosomal preparations and the effects of various agents on the labeling. The results 3 confirmed the higher laC content of dopamine and also led us to suggest that the tyrosme intermediate that is formed from phenylalanine in brain tissue is not freely miscible with the endogenous free tyrosine. The lack of free miscibility may possibly be due to some sort of compartmentation of the tyrosine formed, but the reason for such compartmentation is not clear at the present time. Question may be asked however if any other catecholamine related compounds besides tyrosine, may have a similar lack of miscibility. It may also be asked if this property of tyrosine stems from the fact that phenylalanine-derived tyrosine is the product and substrate of the same enzyme7,11, since such a situation may conceivably lead to a facilitative localization or compartmentation. In an attempt to answer these questions we have now examined if DOPA, the product of tyrosine hydroxylation, may exhibit any compartmentation like that of tyrosine. The results may possibly throw some light on the mechanism of compartmentation since DOPA, unlike tyrosine, may not be the product and substrate of the same enzyme. For the present experiments, we have incubated [3H]DOPA as cosubstrate with either [14C]phenylalanine or [laC]tyrosine and subsequently compared the double labeling of DOPA m the incubation mixture with that of dopamine. MATERIALS AND METHODS
Animals Female Wistar rats (150-175 g) were supplied by Carworth, New City, N.Y. The animals were fasted overnight before the day of the experiment.
Labeled compounds Phenylalanine uniformlylabeled with 14C(L-phenylalanine-U-14C; specific radioactivity 350-400 mCi/mmole), [14C]dopamine (3,4-dihydroxyphenylethylamine, HBr (ethyl-l-14C); specific radioactivity 9.28 mCi/mmole) and [14C]tyrosine (Ltyrosine-U-14C; specific radioactivity 404 mCi/mmole) were obtained from New England Nuclear, Boston, Mass. Tyrosine labeled with 3H (L-p-hydroxyphenyl (alanine-2,3-3H); specific radioactivity 15600 mCi/mmole) and [3H]DOPA (L-3.4dihydroxyphenyl (2,3-3H) alanine; specific radioactivity 2400 mCi/mmole) was purchased from Amersham Searle, Arlington Heights, Ill. All the labeled catecholic compounds were purified at regular intervals by absorption on acid washed alumina and subsequent elution with 0.5 N acetic acid. The high specific radioactivity of tritiated DOPA was reduced by adding cold L-DOPA and the labeled compound was maintained at the lower specific activity. The radiochemicat purity of labeled phenylalanine and tyrosine was checked periodically by subjecting them to our analytical procedures as employed for the separation of the enzyme incubation products. Such blank analysis usually showed very low (2-3 time background count
593 rate) radioactivity in various fractions containing the compounds of our interest.
Incubation methods The methods of incubation were essentially same as described beforel,L Each incubation mixture contained tissue preparation (equivalent to 20 mg wt. brain tissue), mercaptoethanol (0.05 M), pargyline hydrochloride (0.4 mM), sodium phosphate buffer of indicated pH and the labeled substrates. The substrate concentrations/ specific radioactivities were as mentioned in the Results. No other addition of any kind, cofactor or otherwise, was made to the incubation mixture. For preparing the crude synaptosomal-mitochondrial fraction from the caudate nucleus region, the tissue sample was homogenized (10~) in ice-cold 0.32 M sucrose containing 10 micromolar calcium chloride. The sucrose homogenate was spun once at 27,000 x g for 15 min and the sediment was gently resuspended in sodium phosphate buffer before incubation. The synaptosomal preparation in phosphate buffer was incubated with suitably labeled DOPA as the cosubstrate with either labeled phenylalanine or labeled tyrosine (see Results). The mixture was shaken at 37 °C in open test tubes before the addition of an equal volume of ice cold 0.8 N perchloric acid to stop the reaction. In some of the experiments, the synaptosomal particles were separated from the incubation medium at the termination of the incubation period. For this purpose, 2.0 ml of ice-cold mixture identical in composition with that of the incubation medium was added to the incubation mixture to stop the reaction. The particulates were immediately separated from the medium by pouring the incubation mixture on an 0.8 nm size Millipore filter under suction. The collected particulates were further washed by an additional 2.0 ml of ice-cold medium and the filter was then dropped in 2.0 ml of 0.4 N perchloric acid containing 0.1 ~ Triton X-100. The separated incubation medium was mixed with an equal volume of 0.8 N perchloric acid. The acid extracted samples from either the separated particulates and medium or the whole incubation mixtures were then analyzed as mentioned below. The results reported are the averages of the indicated number of experimental values. Calculation of the standard error of the mean (4- S.E.M.) and the probability (P) values were according to Snedecor and Cochran 12.
Analytical methods The analysis of the incubation mixture following 2 × extraction with 0.4 N perchloric acid was done essentially as described before1, z. The procedure involved, after the addition of 20 #g each of DOPA and dopamine carrier, initial fractionation on Dowex-50 (Na +) ion-exchange column to separate the acids, neutrals (amino acids) and the amines. The column effluent containing the acidic compounds was adjusted to pH 8.4 after adding 100 mg of ascorbic acid. The 3,4-dihydroxyphenylacetic acid (DOPAC) present in this fraction was absorbed onto acid washed alumina which was washed with water and 0.2 M sodium acetate solution before the elution of DOPAC by slowly percolating 2.5 ml of 1 N H2SO4 through the alumina contained in a column. The sulfuric acid eluate was then adjusted to pH 2 after saturating with sodium chloride and Dopac was extracted into 6.0 ml of ethyl acetate by thor-
594 ough shaking on a vortex mixer. The organic layer was mixed with 15 ml of Aquasol (New England Nuclear, Boston, Mass ) scintillation fluid for radioactivity determination. The dopamine present m the amine fraction from the Dowex-50 (Na Y) ionexchange column was further purified by acid alumina absorption and elutlon with 0.5 N acetic acid as described before1, 2 for the catecholammes. Usually the acetic acid eluate was assayed for the total radioactivity unless subjected to paper chromatography 1 to detect the presence of any labeled norepinephrine. The neutral fraction from the initial analysis of the incubation mixture extract on Dowex-50 (Na +) column contained DOPA, tyrosine and phenylalanine. Following the addition of 0.5 ml of 0.5 M sodium phosphate buffer (pH 7.0) the pH of this neutral fraction was adjusted to 7.0. Crude kidney DOPA decarboxylase (E.C. 4.1.1.26) preparation" was added to this fraction which was then incubated for 30 mm at 37 °C for the complete decarboxylation of DOPA. The mixture was then acidified to pH 2 and fractionated again on Dowex-50 (Na +) column to separate the neutrals, phenylalanine and tyrosine, from the amine fraction. Dopamine present in the amine fraction was further purified and assayed for radioactivity as described above. The radioactivity in this fraction therefore ~s that of DOPA present in the incubation mixture assayed as dopamme The ion-exchange and paper chromatographic methods for the separation and purification of phenylalanme and tyrosme before radioactivity assay have been descmbed before 1. The paper chromatographic system employed completely separates tyrosine and phenylalanine and any DOPA that may be present. The analytical recoveries were determined by adding known labeled compounds to perchloric acid extract of brain tissue and carrying through the complete analytical procedures. The recoveries were 55 ~o for dopamine, 42 % for DOPA and 50 o/,, for tyrosine as determined from a large number of such analyses. No correction for recovery has been apphed to any of the data in Results. The determinations of the endogenous concentrations of phenylalanine and tyrosine were performed as mentioned before 2,z by fluor~metry.
Assay of the double labehng The method was as described previously 2 for the assay of double labeling by Beckman liquid scintillation counter (model 100 C). The radioactivities of various labeled substrates were so chosen that the double labeling of the products could be determined with mimmum error due to spill. The efficiencies of isotope counting in the compounds after the analytical separations were checked by internal standards. The per cent efficiencies were 62% for 14C and 31 ~ for SH. Isotopes were counted long enough to reach a low statistical error, usually 3 % or less. Following the determination of the disintegrations per min of each isotope, the isotope ratios were calculated. This ratio was defined as the isotope in any labeled substrate expressed as a percentage of the label in the cosubstrate which appears later in the well established metabolic sequence of phenylalanine --+ tyrosine --+ DOPA - ~ dopamine - ~ DOPAC. By this definition, if the isotope ratio in any compound is observed to be higher than that in its precursor, some sort of preferential isotope incorporation
595 TABLE I DOPA AND DOPAMINE(DA) SYNTHESISFROM THE SUBSTRATEMIXTURESBY CAUDATEPARTICULATES The caudate nucleus particulates were prepared and suspended in pH 6.5 phosphate buffer as described in the Materials and Methods. The incubation mixture contained particulates from 20 nag caudate, 0.05 M 2-mercaptoethanol, 0.4 rnM pargyline hydrochloride (MAO inhibitor) and 0.127 M sodium phosphate in a final volume of 0.66 ml. The incubation was for 30 min at 37 °C. The substrate concentrations (mlcromolar) and specific radioactivities (nCi/nmole) respectively were: [14C]phenylalanine, 5.4 and 30.1 ; [14C]tyrosine, 5.2 and 29.4; [SH]DOPA, 1.8 and 18.9. The results are expressed as mean plus minus standard error of the mean. The probability values were calculated by 2-tailed t-tests. For the definition of the isotope ratio see Materials and Methods. Substrate mixture
Isotope ratio (14C/3H × 100)
Products formed (nmole/g/h)
[14C]-
[14C]-
[3HI-
DOPA
DA
DA
[14C]phenylalanine and pH]DOPA 0.53 4- 0.02 (N = 4) [14C]tyrosine and 2.3 4- 0.07 [aH]DOPA (N = 4) (2.9 4- 0.1)*
4.33 4- 0.12 23.0 ± 0.7
R(DOPA)
R(DA)
2.0 4- 0.1
30.1 -4- 0.5 P < 0.001
10.2 -I- 0.6 21.8 4- 0.9
8.8 4- 0.2
73.0 4- 1.7 P < 0.001
(9.9 4- 0.4) (17.0 4- 0.2) (10.9 4- 0.5).
(106.0 q- 3.1) (P < 0.001)
* The values within parentheses are from experiments employing synaptosomal-mitochondrial (P2) suspension prepared according to Whittaker TM and washed once. In these experiments [SH]DOPA was 16.0 nCi/nmole.
may be indicated. All isotope ratios are indicated by the symbol R with the compound within parenthesis i.e. R(DOPA). RESULTS
Double labeling of DOPA and dopaminefrom the labeled substrate mixtures The double labeling data of DOPA and dopamine, R(DOPA) and R(DA) values, following the incubations, are summarized in Table I. The results show that the caudate particulates formed DOPA and dopamine from [14C]phenylalanine and dopamine from [aH]DOPA. The results also show that the R(DA) value was 15 times larger (P < 0.001) than the R(DOPA) value. In our experiments with the substrate mixture of [14C]tyrosine and [3H]DOPA, considerable difference was again observed between R(DOPA) and R(DA); the R(DA) value was 8.3 times the R(DOPA) value. The results also indicate that of the total [x4C]DOPA formed, from either [4C]phenylalanine or [14C]tyrosine, 82-89 % was decarboxylated to dopamine. The double labeling of dopamine and DOPA C by caudate particulates Our observed differences between the double labeling of DOPA and that of dopamine (Table I) prompted us to investigate the double labeling of another pair of closely related compounds. We compared the double labeling of dopamine with
596 TABLE II DOPAMINE AND D O P A C
SYNTHESIS FROM THE SUBSTRATE MIXTURES BY CAUDATE PARTICULATES
The experimental conditions were as described for Table I excepting for the omission of pargyhne hydrochloride from the incubation mixtures. The substrate concentrations (micromolar) and the specific radloactwttles (nC1/nmole) respectively were: [aH]tyrosme, 5.3 and 23.4; [aH]DOPA, 1 9 and 18.9; [14C]DA, 1.6 and 4 I For the definltmn of the isotope ratio see Materials and Methods Substrate mi.~tm e
[3H]DOPA and [14C]DA (N = 4) [aH]tyrosineand [14C]DA (N = 4)
Products formed (nmole/g/h)
L~otope ratio (aH/~aC ~ 100) - - ,r14C]DOPAC R ( D A ) R(DOPAC)
3HJDA
~H]DOPAC
17.7 ± 0.2
1.4 ± 0.04
7.2 ± 0.1
80.0 ± 0.8
88.0 ± 1 3
17.9±0.4
0.91 ~0.02
10.5 ±0.5
1170±2.7
50.0± 1.9
that of DOPAC, a major metabolite of brain dopaminO ° formed by the action of MAO (E.C. 1.4.3.4). For these experiments pargyline was omitted from the incubation mixtures. The results (Table II) show that the isotope ratio in DOPAC formed from the substrate mixture of [14C]dopamine and [3H]DOPA was 88.0 and it was only slightly higher than the ratio (80.0) in dopamine. These data do not indicate a large difference between the ratio in dopamine and that in DOPAC. Following the incubation of the [14C]dopamine [aH]tyrosine cosubstrates, the DOPAC isotope ratio was 50.0 and it was clearly lower than dopamine ratio (117.0). S u b s t r a t e concentrations a n d the isotope ratios in D O P A a n d d o p a m i n e
Influence of substrate concentration on enzymatic product formation is wellknown. We have considered that the isotope ratio in dopamine and also the difference between R(DA) and R(DOPA) values may be altered by any changes in the concentrations of [14C]tyrosine and [aH]DOPA cosubstrates. The results of the experiments at different substrate concentrations are summarized in Table III. It may be seen that with the increases of [aH]DOPA and [14C]tyrosine concentrations, the respective products increased. However, the maximum hydroxylation was probably reached at or below 10.7 # M tyrosine. It should be pointed out that for these incubations, the quantity of isotope added to the incubation mixtures was kept constant while the substrate concentrations were increased. It may be seen from the results that the changes of substrate concentrations and therefore specific radioactivities, affected the isotope ratios. At 1.8 /~M [aH]DOPA concentrations, R ( D A ) decreased from 290.0 to 68.8 following tyrosine concentrations increase from 1.9 to 10.7 pM. However, R(DOPA) value decreased concomitantly and remained much lower than R(DA). The marked difference between the ratios remained so when [3H]DOPA concentration was raised from 1.8 to 17.5 p M at constant 5.3/~M tyrosine. In all of these incubations, the R(DA) values were between 10.6 to 13.7 times the ratios in DOPA.
COSUBSTRATE CONCENTRATIONS AND THE ISOTOPE RATIOS IN THE PRODUCTS D O P A
AND DOPAMINE
18.0 4- 1.0 18.2 q- 0.8 23.3 4- 0.8
11.2 4- 0.1 6.8 4- 0.2 10.4 4- 0.4
27.9 4- 0.9
24.8 4- 1.3
26.9 4- 1.4
189.0 4- 1.9
26.3 4- 1.3
ff3H]DA
6.4 4- 0.3
25.3 4- 0.5
12.9 4- 0.2
11.5 4- 0.9
R(DOPA)
P < 0.001
P < 0.001
e < 0.001
P < 0.001
Isotope ratio (14C/3H X 100)
* The values within parentheses are the synaptosomal uptake (nmoles/g/10 mm) of the substrates at the indicated concentrations.
5.3 (21.0) 1.9 (9.0) 10.7 (42.3) (65.0at20 x 10-6M)
1.8 (8.6)* 17.5 (79.7) 1.8 (8.5) 1.8 (8.3)
5.3
9.5 4- 0.3
{14C]DOPA
{aH]DOPA
{14CJtyrosine
{I~C]DA
Products formed ( nmole /g/ h)
Substrate concentration ( I~M )
68.8 4- 3.0
290.0 4- 2.7
137.0 4- 4.9
158.0 4- 5.5
R(DA)
Caudate nucleus particulates from 24 mg tissue were prepared and suspended in p H 6.0 phosphate buffer as described in the Materials and Methods. The incubation mixture contained mercaptoethanol, pargyline and phosphate as mentioned in Table I. Each incubation mixture also contained t01.6 nCi of [14C]tyrosine and 22.7 nCl of [3H]DOPA in a final volume of 0.66 ml. The incubation was for 10 min at 37 °C.
[14C]TYROSINE AND [ 3 H ] D O P A
TABLE III
598 TABLE IV ['~H]DOPA
INCUBATION OF CAUDATE PARTICULATES WITH [t4C]TYROSINE
COSUBSTRATES
DOPA
AND
DOPAMINE ISOTOPE RATIOS IN THE PARTICULATES AND MEDIUM
The substrate concentrations (mlcromolar) and specific radloactJvlties (nG/nmole) respectively ~vere: [14C]tyrosme, 5.3 and 28.0; [aH]DOPA, 9.1 and 3.9. In all other respects, the incubation method was same as that described in Table III. Following the lncubahon the particulates were separated from the Incubation medium on Milhpore filters as described in the Materials and Methods. .
.
Fraction analyzed
Particulates
.
.
.
.
.
.
.
.
.
.
.
.
.
.
f 14Cf-
DOP14
DA
1.3 ± 0 1
4 5 zk 0 2
.
.
.
.
.
.
.
.
Isotope ratio (14C/3H x 100)
Productsformed (nmole/g/h)
~/14C7-
.
,73H~DA
R(DOPA)
22.6 ± 1.1
12.2 ± 0.4
R(DA)
147.0 ± 1 9 P f 0.001
Medium
9.6±04
16.8±0.7
88.0=t=5.5
13.5±0.1
1392 ~ 2.7 P (0001
DOPA and dopamine isotope ratios in the synaptosomes
The enzymes tyrosine hydroxylase and DOPA decarboxylase have been observed 4,s in the caudate nucleus synaptosomal particles. The formation o f DOPA and dopamine from the tyrosine and DOPA cosubstrates in our present experiments may occur in the particulates following the uptake of the substrates. Although studies 5,9 with synaptosomes suggest rapid uptake of exogenous amino acids and their quick equilibration with the endogenous pool, we wished to compare the isotope ratios in the particulates where the hydroxylation and decarboxylation may actually occur. For these experiments, caudate synaptosomal preparations were obtained as described in the Materials and Methods. Following their incubation with the substrate mixture of [14C]tyrosine and [SH]DOPA, the synaptosomal particulates were quickly separated from the medium by filtering on 0.8 nm Millipore filter (see Materials and Methods). Each separated fraction, particulates and medium, was then analyzed and the isotope ratios determined. The results (Table IV) indicate that the products formed were distributed between the medium and the particulates and the latter fraction contained 12%, 21% and 2 0 ~ respectively of [t4C]DOPA, [14C]dopamine and [SH]dopamine. Incidentally, there were sufficient radioactivities in these samples to allow less than 5 % counting error. The results also show that in the particulates, as well as in the medium, the R(DA) value was much larger then R(DOPA) and in fact 10.3-12.0 times larger. Our time study (Fig. 1). also reveals that the difference between the isotope ratios in the synaptosomal particles was significantly (P < 0.001 ) large during the incubation periods ranging from 10 to 45 min. DISCUSSION
The results in Table I show that both DOPA and dopamine are formed from [14C]phenylalanine and [14C]tyrosine following the incubation with the caudate
599
150
125
0
I00
m ul 0
75
N 0
~
5o
25
I0 M I N U T E S
20
30
45
p (0.001
Fig. 1. Temporal changes of intrasynaptosomal R(DOPA) and R(DA) values. For these incubations the substrate concentrations (micromolar) and specificradioactivities (nCi/nmole) respectively were: [14C]tyrosine,5.3 and 29.4; [SH]DOPA, 1.8 and 18.9. In all other respects, the experiments were as described for Table IV. preparation. Paper chromatographic separation followed by radioactivity assay carried out with a number of samples indicated only a minor contamination of dopamine by labeled norepinephrine, usually 10% or less, as we have reported 1 before. The lack of any significant norepinephrine formation permitted us to use the side chain tritium-labeled tyrosine in some of our experiments with no loss of radioactivity. We have employed relatively crude preparation of the synaptosomal-mitochondrial fraction for most of our studies. However, for some of our experiments we have prepared the fraction (P2) according to Whittaked s, washed once and incubated with [14C]tyrosine [SH]DOPA cosubstrates. The results (Table I) are essentially indistinguishable from those data from incubation of the relatively crude fractions (Table I). It may be seen from the results in Table I that the isotope ratio in dopamine formed by caudate preparations differs significantly from the ratio in DOPA. The ratio in dopamine was clearly higher than R(DOPA) from the incubation with [14C]phenylalanine in the presence of [SH]DOPA. If the [14C]DOPA product of
600 [14C]phenylalanine hydroxylatlon is freely exchangeable with the [3H]DOPA in the medium, one may rather expect a lower isotope ratio m dopamine. The [3H]DOPA present initially in the medium was being continuously decarboxylated from the very start of the incubation before any [14C]DOPA could be formed. Similarly, if the [14C]DOPA gradually formed from [l'4C]tyrosme is freely exchangeable with the [3H]DOPA present in the medium, a R(DA) value lower than R(DOPA) should result. The data (Table 1) indicate that from [14C]tyrosine [3H]DOPA cosubstrate mixture as well, R(DA) is higher than R(DOPA). In some experiments we have switched isotopes, i.e., have employed [3H]tyrosine as cosubstrate with [14C]DOPA. The results ([3H]DOPA, 4.4 ! 0.1 nmole/g/h; [3H]DA, 13.5 ± 0.1; [14C]DA, 23.3 :~0.3; R(DA), 1227 0 ~ 9.7 and R(DOPA), 134.0 ± 2.7) show again that R(DA) was much higher than R(DOPA). Since free exchange between the newly formed [14C]DOPA and exogenous [3H]DOPA does not appear to take place, some sort of compartmentation of the newly formed DOPA is indicated. The higher isotope ratio in dopamine also suggests a preferential decarboxylation of [14C]DOPA formed from either phenylalanine or tyrosine. In comparison with our findings on DOPA, the data on the oxidation of dopamine to DOPAC and the corresponding isotope ratios (Table II) do not clearly suggest any compartmentation of dopamine. When we chose [3H]DOPA as the precursor of newly formed dopamine m the presence of added [14C]dopamme, the isotope ratios in DOPAC and dopamine did not differ markedly. The isotope ratio m DOPAC was clearly lower than in dopamine from the incubation of the substrate mixture of [aH]tyrosine and [14C]dopamine. It is however possible that the exogenous dopamme and that formed in situ are not freely exchangeable and the respectlve oxidations by MAO proceeds at different rates. The reason for the compartmentation of DOPA ~s not clear at the present time but our results in Table Ill and Table IV indicate some basic facts about the compartmentatlon. Tyrosine and DOPA are expected to interact during synaptosomal uptake and subsequent enzymatic reactions. The data on the uptake of tyrosine and DOPA cosubstrates (Table IlI) show that increases of uptake resulted from the elevation of substrate concentrations. The increase of DOPA uptake from 8.3 to 79.7 nmoles/g/10 rain and that of tyrosine uptake from 9.0 to 42.3 nmoles/g/10 min (Table lII) increased the respective product formations and led to new values of R(DOPA) and R(DA). However, R(DA) values remained significantly (P < 0.001) higher (10.6-13.7 times) than that of R(DOPA) at all the substrate concentrations tested. It appears therefore that whatever may be the mechanism of the compartmentation, it is not an artifactual effect of the substrate concentrations chosen or the resultant uptakes. The results in Table IV show that the isotope ratio in the particulates, whether m DOPA or in dopamine, was very close to that in the medium. The results of analysis of the entire incubation mixture may therefore accurately reflect the values m separated synaptosomes. Since the R(DOPA) and R(DA) values from the synaptosomes differed just as significantly, the preferential decarboxylation of newly formed DOPA may occur intrasynaptosomally. To sum up, it appears that DOPA that is formed in situ by the hydroxylation of its precursors may not be freely exchangeable with exogenous DOPA because
601 o f c o m p a r t m e n t a t i o n and the newly formed D O P A m a y be preferentially decarboxylated. One o f several alternative explanations for these p h e n o m e n a is that, in the synaptosomes, the hydroxylating and the decarboxylating enzymes are organized advantageously in close proximity. A l t h o u g h such an organization has been proposed for the enzymes o f serotonin synthesis 6, our present data do not necessarily prove such an organization o f tyrosine hydroxylase and D O P A decarboxylase enzymes. Finally, the c o m p a r t m e n t a t i o n o f D O P A and that o f tyrosine as indicated by our results ~,a appear rather similar. I f indeed similar mechanisms underlie both the cases, the c o m p a r t m e n t a t i o n of tyrosine is not due to its being the p r o d u c t and substrate o f the same enzyme. Individually characteristic mechanism o f c o m p a r t m e n t a t i o n is however entirely possible. ACKNOWLEDGMENT
This work was supported by the Department of Mental Hygiene, State of New York.
REFERENCES 1 BAGCHI, S. P., AND ZARYCKI, E. P., Hydroxylation of phenylalanine by various areas of brain
in vitro, Biochem. Pharmacol., 21 (1972) 584-589. 2 BAGCHI,S. P., ANDZARYCKI,E. P., Formation of catecholamines from phenylalanine in brain - effects of chlorpromazine and Catron, Biochem. Pharmacol., 22 (1973) 1353-1368. 3 BAOCHI,S. P., AND ZARYCKI,E. P., Catecholamine formation in brain from phenylalanine and tyrosine: effects of psychotropic drugs and other agents, Biochem. Pharmacol., 24 (1975) 13811390. 4 FAHN, S., RODMAN,J. S., AND C6T~, L. J., Association of tyrosine hydroxylase with synaptic vesicles in bovine nucleus, J. Neurochem., 16 (1969) 1293-1300. 5 GRAHAME-SMITH,D. G., AND PARHTT, A. G., Tryptophan transport across the synaptosomai membrane, J. Neurochem,, 17 (1970) 1339-1353. 6 ICHIYAMA,A., NAKAMURA,S., NISHmUKA,Y., AND HAY.MSHI,O., Tryptophan-5-hydroxylase in mammalian brain, Advanc. Pharmacol., 6A (1968) 5-17. 7 IKEDA,M., LEvrrr, M., ANDUDENFREND,S., Phenylalanine as substrate and inhibitor of tyrosine hydroxylase, Arch. Biochem. Biophys., 120 (1967) 420-427. 8 MCGEER,P. L., BAGCHI,S. P., AND McGEER, E. G., Subcellular localization of tyrosine in beef caudate nucleus, Life Sci., 4 (1965) 1859-1867. 9 PETERSON,N. A., AND RAGHUPATHY,E., Characteristics of amino acid accumulation by synaptosomal particles isolated from rat brain, J. Neurochem., 19 (1972) 1423-1438. l0 RIDDELL,D., AND SZERB, J. C., The release in rive of dopamine synthesized from labelled precursors in the caudate nucleus of the cat, J. Neurochem., 18 (1971) 989-1006. 11 SHIMAN,R., AK]NO, M., AND KAUFMAN,S., Solubihzation and partial purification of tyrosine hydroxylase from bovine adrenal medulla, J. biol. Chem., 246 (1971) 1330-1340. 12 SNEDECOR,G. W., AND COCHRAN,W. G., Statistical methods, Iowa State Univ. Press, Ames, Iowa, 1967. 13 WmTTAKER,V. P., Methods of Neurochemistry, Vol. 2, Dekker, New York, 1972.
591
© ElsevierScientificPublishing Company,Amsterdam- Printed in The Netherlands
DECARBOXYLATION OF NEWLY FORMED DOPA BY CAUDATE NUCLEUS SYNAPTOSOMAL PARTICLES
SAKTI P. BAGCHI* ANDTHOMAS M. SMITH Research Division, Marcy Psychiatric Center, Marcy, N.Y. 13403 (U.S.A.)
(Accepted October 4th, 1975)
SUMMARY
The present study compares dopamine formation from two different sources of DOPA: preformed and added to the medium and that newly formed by the hydroxylations of phenylalanine or tyrosine. Synaptosomal preparations from rat brain caudate nucleus were incubated with all-labeled DOPA mixed as cosubstrate with either p4C]phenylalanine or [14C]tyrosine. Following the incubation, DOPA and dopamine were separated and their isotope ratios (isotope in phenylalanine (tyrosine) × 100/isotope in DOPA cosubstrate) were determined and compared. The results show that the ratio in dopamine was not only different from that in DOPA but was in fact 8.3-15.0 times higher. Although the absolute values of the isotope ratios were affected by any changes of the cosubstrate concentrations, the ratio in dopamine was found to be higher than that in DOPA at all the substrate concentrations tested. When the synaptosomes were separated post-incubation from the medium and analyzed, the ratio in dopamine was also found to be higher than that in DOPA. Also, the large difference between the intrasynaptosomal ratios persisted throughout the incubation period ranging from 10 to 45 min. The results suggest that the DOPA which is newly formed by the hydroxylations, (a) may not be freely exchangeable with added DOPA because of compartmentation and (b) may be preferentially decarboxylated to dopamine.
INTRODUCTION During the course of our investigation of phenylalanine hydroxylation by brain tissue, we incubated caudate nucleus homogenate with [14C]phenylalanine mixed * To whom correspondence should be addressed at: Rockland Research Institute, Building 37, Orangeburg, New York, N.Y. 10962, U. S.A.
592 with [3H]tyrosine as the cosubstrate. The results 2 indicated that the isotope ratio (laC/3H / 100) in the dopamine formed was higher than that in the tyrosine isolated. In an attempt to clarify the reason for the higher 14C content of dopamine we further investigated the double labeling of dopamine and tyrosine by caudate synaptosomal preparations and the effects of various agents on the labeling. The results 3 confirmed the higher laC content of dopamine and also led us to suggest that the tyrosme intermediate that is formed from phenylalanine in brain tissue is not freely miscible with the endogenous free tyrosine. The lack of free miscibility may possibly be due to some sort of compartmentation of the tyrosine formed, but the reason for such compartmentation is not clear at the present time. Question may be asked however if any other catecholamine related compounds besides tyrosine, may have a similar lack of miscibility. It may also be asked if this property of tyrosine stems from the fact that phenylalanine-derived tyrosine is the product and substrate of the same enzyme7,11, since such a situation may conceivably lead to a facilitative localization or compartmentation. In an attempt to answer these questions we have now examined if DOPA, the product of tyrosine hydroxylation, may exhibit any compartmentation like that of tyrosine. The results may possibly throw some light on the mechanism of compartmentation since DOPA, unlike tyrosine, may not be the product and substrate of the same enzyme. For the present experiments, we have incubated [3H]DOPA as cosubstrate with either [14C]phenylalanine or [laC]tyrosine and subsequently compared the double labeling of DOPA m the incubation mixture with that of dopamine. MATERIALS AND METHODS
Animals Female Wistar rats (150-175 g) were supplied by Carworth, New City, N.Y. The animals were fasted overnight before the day of the experiment.
Labeled compounds Phenylalanine uniformlylabeled with 14C(L-phenylalanine-U-14C; specific radioactivity 350-400 mCi/mmole), [14C]dopamine (3,4-dihydroxyphenylethylamine, HBr (ethyl-l-14C); specific radioactivity 9.28 mCi/mmole) and [14C]tyrosine (Ltyrosine-U-14C; specific radioactivity 404 mCi/mmole) were obtained from New England Nuclear, Boston, Mass. Tyrosine labeled with 3H (L-p-hydroxyphenyl (alanine-2,3-3H); specific radioactivity 15600 mCi/mmole) and [3H]DOPA (L-3.4dihydroxyphenyl (2,3-3H) alanine; specific radioactivity 2400 mCi/mmole) was purchased from Amersham Searle, Arlington Heights, Ill. All the labeled catecholic compounds were purified at regular intervals by absorption on acid washed alumina and subsequent elution with 0.5 N acetic acid. The high specific radioactivity of tritiated DOPA was reduced by adding cold L-DOPA and the labeled compound was maintained at the lower specific activity. The radiochemicat purity of labeled phenylalanine and tyrosine was checked periodically by subjecting them to our analytical procedures as employed for the separation of the enzyme incubation products. Such blank analysis usually showed very low (2-3 time background count
593 rate) radioactivity in various fractions containing the compounds of our interest.
Incubation methods The methods of incubation were essentially same as described beforel,L Each incubation mixture contained tissue preparation (equivalent to 20 mg wt. brain tissue), mercaptoethanol (0.05 M), pargyline hydrochloride (0.4 mM), sodium phosphate buffer of indicated pH and the labeled substrates. The substrate concentrations/ specific radioactivities were as mentioned in the Results. No other addition of any kind, cofactor or otherwise, was made to the incubation mixture. For preparing the crude synaptosomal-mitochondrial fraction from the caudate nucleus region, the tissue sample was homogenized (10~) in ice-cold 0.32 M sucrose containing 10 micromolar calcium chloride. The sucrose homogenate was spun once at 27,000 x g for 15 min and the sediment was gently resuspended in sodium phosphate buffer before incubation. The synaptosomal preparation in phosphate buffer was incubated with suitably labeled DOPA as the cosubstrate with either labeled phenylalanine or labeled tyrosine (see Results). The mixture was shaken at 37 °C in open test tubes before the addition of an equal volume of ice cold 0.8 N perchloric acid to stop the reaction. In some of the experiments, the synaptosomal particles were separated from the incubation medium at the termination of the incubation period. For this purpose, 2.0 ml of ice-cold mixture identical in composition with that of the incubation medium was added to the incubation mixture to stop the reaction. The particulates were immediately separated from the medium by pouring the incubation mixture on an 0.8 nm size Millipore filter under suction. The collected particulates were further washed by an additional 2.0 ml of ice-cold medium and the filter was then dropped in 2.0 ml of 0.4 N perchloric acid containing 0.1 ~ Triton X-100. The separated incubation medium was mixed with an equal volume of 0.8 N perchloric acid. The acid extracted samples from either the separated particulates and medium or the whole incubation mixtures were then analyzed as mentioned below. The results reported are the averages of the indicated number of experimental values. Calculation of the standard error of the mean (4- S.E.M.) and the probability (P) values were according to Snedecor and Cochran 12.
Analytical methods The analysis of the incubation mixture following 2 × extraction with 0.4 N perchloric acid was done essentially as described before1, z. The procedure involved, after the addition of 20 #g each of DOPA and dopamine carrier, initial fractionation on Dowex-50 (Na +) ion-exchange column to separate the acids, neutrals (amino acids) and the amines. The column effluent containing the acidic compounds was adjusted to pH 8.4 after adding 100 mg of ascorbic acid. The 3,4-dihydroxyphenylacetic acid (DOPAC) present in this fraction was absorbed onto acid washed alumina which was washed with water and 0.2 M sodium acetate solution before the elution of DOPAC by slowly percolating 2.5 ml of 1 N H2SO4 through the alumina contained in a column. The sulfuric acid eluate was then adjusted to pH 2 after saturating with sodium chloride and Dopac was extracted into 6.0 ml of ethyl acetate by thor-
594 ough shaking on a vortex mixer. The organic layer was mixed with 15 ml of Aquasol (New England Nuclear, Boston, Mass ) scintillation fluid for radioactivity determination. The dopamine present m the amine fraction from the Dowex-50 (Na Y) ionexchange column was further purified by acid alumina absorption and elutlon with 0.5 N acetic acid as described before1, 2 for the catecholammes. Usually the acetic acid eluate was assayed for the total radioactivity unless subjected to paper chromatography 1 to detect the presence of any labeled norepinephrine. The neutral fraction from the initial analysis of the incubation mixture extract on Dowex-50 (Na +) column contained DOPA, tyrosine and phenylalanine. Following the addition of 0.5 ml of 0.5 M sodium phosphate buffer (pH 7.0) the pH of this neutral fraction was adjusted to 7.0. Crude kidney DOPA decarboxylase (E.C. 4.1.1.26) preparation" was added to this fraction which was then incubated for 30 mm at 37 °C for the complete decarboxylation of DOPA. The mixture was then acidified to pH 2 and fractionated again on Dowex-50 (Na +) column to separate the neutrals, phenylalanine and tyrosine, from the amine fraction. Dopamine present in the amine fraction was further purified and assayed for radioactivity as described above. The radioactivity in this fraction therefore ~s that of DOPA present in the incubation mixture assayed as dopamme The ion-exchange and paper chromatographic methods for the separation and purification of phenylalanme and tyrosme before radioactivity assay have been descmbed before 1. The paper chromatographic system employed completely separates tyrosine and phenylalanine and any DOPA that may be present. The analytical recoveries were determined by adding known labeled compounds to perchloric acid extract of brain tissue and carrying through the complete analytical procedures. The recoveries were 55 ~o for dopamine, 42 % for DOPA and 50 o/,, for tyrosine as determined from a large number of such analyses. No correction for recovery has been apphed to any of the data in Results. The determinations of the endogenous concentrations of phenylalanine and tyrosine were performed as mentioned before 2,z by fluor~metry.
Assay of the double labehng The method was as described previously 2 for the assay of double labeling by Beckman liquid scintillation counter (model 100 C). The radioactivities of various labeled substrates were so chosen that the double labeling of the products could be determined with mimmum error due to spill. The efficiencies of isotope counting in the compounds after the analytical separations were checked by internal standards. The per cent efficiencies were 62% for 14C and 31 ~ for SH. Isotopes were counted long enough to reach a low statistical error, usually 3 % or less. Following the determination of the disintegrations per min of each isotope, the isotope ratios were calculated. This ratio was defined as the isotope in any labeled substrate expressed as a percentage of the label in the cosubstrate which appears later in the well established metabolic sequence of phenylalanine --+ tyrosine --+ DOPA - ~ dopamine - ~ DOPAC. By this definition, if the isotope ratio in any compound is observed to be higher than that in its precursor, some sort of preferential isotope incorporation
595 TABLE I DOPA AND DOPAMINE(DA) SYNTHESISFROM THE SUBSTRATEMIXTURESBY CAUDATEPARTICULATES The caudate nucleus particulates were prepared and suspended in pH 6.5 phosphate buffer as described in the Materials and Methods. The incubation mixture contained particulates from 20 nag caudate, 0.05 M 2-mercaptoethanol, 0.4 rnM pargyline hydrochloride (MAO inhibitor) and 0.127 M sodium phosphate in a final volume of 0.66 ml. The incubation was for 30 min at 37 °C. The substrate concentrations (mlcromolar) and specific radioactivities (nCi/nmole) respectively were: [14C]phenylalanine, 5.4 and 30.1 ; [14C]tyrosine, 5.2 and 29.4; [SH]DOPA, 1.8 and 18.9. The results are expressed as mean plus minus standard error of the mean. The probability values were calculated by 2-tailed t-tests. For the definition of the isotope ratio see Materials and Methods. Substrate mixture
Isotope ratio (14C/3H × 100)
Products formed (nmole/g/h)
[14C]-
[14C]-
[3HI-
DOPA
DA
DA
[14C]phenylalanine and pH]DOPA 0.53 4- 0.02 (N = 4) [14C]tyrosine and 2.3 4- 0.07 [aH]DOPA (N = 4) (2.9 4- 0.1)*
4.33 4- 0.12 23.0 ± 0.7
R(DOPA)
R(DA)
2.0 4- 0.1
30.1 -4- 0.5 P < 0.001
10.2 -I- 0.6 21.8 4- 0.9
8.8 4- 0.2
73.0 4- 1.7 P < 0.001
(9.9 4- 0.4) (17.0 4- 0.2) (10.9 4- 0.5).
(106.0 q- 3.1) (P < 0.001)
* The values within parentheses are from experiments employing synaptosomal-mitochondrial (P2) suspension prepared according to Whittaker TM and washed once. In these experiments [SH]DOPA was 16.0 nCi/nmole.
may be indicated. All isotope ratios are indicated by the symbol R with the compound within parenthesis i.e. R(DOPA). RESULTS
Double labeling of DOPA and dopaminefrom the labeled substrate mixtures The double labeling data of DOPA and dopamine, R(DOPA) and R(DA) values, following the incubations, are summarized in Table I. The results show that the caudate particulates formed DOPA and dopamine from [14C]phenylalanine and dopamine from [aH]DOPA. The results also show that the R(DA) value was 15 times larger (P < 0.001) than the R(DOPA) value. In our experiments with the substrate mixture of [14C]tyrosine and [3H]DOPA, considerable difference was again observed between R(DOPA) and R(DA); the R(DA) value was 8.3 times the R(DOPA) value. The results also indicate that of the total [x4C]DOPA formed, from either [4C]phenylalanine or [14C]tyrosine, 82-89 % was decarboxylated to dopamine. The double labeling of dopamine and DOPA C by caudate particulates Our observed differences between the double labeling of DOPA and that of dopamine (Table I) prompted us to investigate the double labeling of another pair of closely related compounds. We compared the double labeling of dopamine with
596 TABLE II DOPAMINE AND D O P A C
SYNTHESIS FROM THE SUBSTRATE MIXTURES BY CAUDATE PARTICULATES
The experimental conditions were as described for Table I excepting for the omission of pargyhne hydrochloride from the incubation mixtures. The substrate concentrations (micromolar) and the specific radloactwttles (nC1/nmole) respectively were: [aH]tyrosme, 5.3 and 23.4; [aH]DOPA, 1 9 and 18.9; [14C]DA, 1.6 and 4 I For the definltmn of the isotope ratio see Materials and Methods Substrate mi.~tm e
[3H]DOPA and [14C]DA (N = 4) [aH]tyrosineand [14C]DA (N = 4)
Products formed (nmole/g/h)
L~otope ratio (aH/~aC ~ 100) - - ,r14C]DOPAC R ( D A ) R(DOPAC)
3HJDA
~H]DOPAC
17.7 ± 0.2
1.4 ± 0.04
7.2 ± 0.1
80.0 ± 0.8
88.0 ± 1 3
17.9±0.4
0.91 ~0.02
10.5 ±0.5
1170±2.7
50.0± 1.9
that of DOPAC, a major metabolite of brain dopaminO ° formed by the action of MAO (E.C. 1.4.3.4). For these experiments pargyline was omitted from the incubation mixtures. The results (Table II) show that the isotope ratio in DOPAC formed from the substrate mixture of [14C]dopamine and [3H]DOPA was 88.0 and it was only slightly higher than the ratio (80.0) in dopamine. These data do not indicate a large difference between the ratio in dopamine and that in DOPAC. Following the incubation of the [14C]dopamine [aH]tyrosine cosubstrates, the DOPAC isotope ratio was 50.0 and it was clearly lower than dopamine ratio (117.0). S u b s t r a t e concentrations a n d the isotope ratios in D O P A a n d d o p a m i n e
Influence of substrate concentration on enzymatic product formation is wellknown. We have considered that the isotope ratio in dopamine and also the difference between R(DA) and R(DOPA) values may be altered by any changes in the concentrations of [14C]tyrosine and [aH]DOPA cosubstrates. The results of the experiments at different substrate concentrations are summarized in Table III. It may be seen that with the increases of [aH]DOPA and [14C]tyrosine concentrations, the respective products increased. However, the maximum hydroxylation was probably reached at or below 10.7 # M tyrosine. It should be pointed out that for these incubations, the quantity of isotope added to the incubation mixtures was kept constant while the substrate concentrations were increased. It may be seen from the results that the changes of substrate concentrations and therefore specific radioactivities, affected the isotope ratios. At 1.8 /~M [aH]DOPA concentrations, R ( D A ) decreased from 290.0 to 68.8 following tyrosine concentrations increase from 1.9 to 10.7 pM. However, R(DOPA) value decreased concomitantly and remained much lower than R(DA). The marked difference between the ratios remained so when [3H]DOPA concentration was raised from 1.8 to 17.5 p M at constant 5.3/~M tyrosine. In all of these incubations, the R(DA) values were between 10.6 to 13.7 times the ratios in DOPA.
COSUBSTRATE CONCENTRATIONS AND THE ISOTOPE RATIOS IN THE PRODUCTS D O P A
AND DOPAMINE
18.0 4- 1.0 18.2 q- 0.8 23.3 4- 0.8
11.2 4- 0.1 6.8 4- 0.2 10.4 4- 0.4
27.9 4- 0.9
24.8 4- 1.3
26.9 4- 1.4
189.0 4- 1.9
26.3 4- 1.3
ff3H]DA
6.4 4- 0.3
25.3 4- 0.5
12.9 4- 0.2
11.5 4- 0.9
R(DOPA)
P < 0.001
P < 0.001
e < 0.001
P < 0.001
Isotope ratio (14C/3H X 100)
* The values within parentheses are the synaptosomal uptake (nmoles/g/10 mm) of the substrates at the indicated concentrations.
5.3 (21.0) 1.9 (9.0) 10.7 (42.3) (65.0at20 x 10-6M)
1.8 (8.6)* 17.5 (79.7) 1.8 (8.5) 1.8 (8.3)
5.3
9.5 4- 0.3
{14C]DOPA
{aH]DOPA
{14CJtyrosine
{I~C]DA
Products formed ( nmole /g/ h)
Substrate concentration ( I~M )
68.8 4- 3.0
290.0 4- 2.7
137.0 4- 4.9
158.0 4- 5.5
R(DA)
Caudate nucleus particulates from 24 mg tissue were prepared and suspended in p H 6.0 phosphate buffer as described in the Materials and Methods. The incubation mixture contained mercaptoethanol, pargyline and phosphate as mentioned in Table I. Each incubation mixture also contained t01.6 nCi of [14C]tyrosine and 22.7 nCl of [3H]DOPA in a final volume of 0.66 ml. The incubation was for 10 min at 37 °C.
[14C]TYROSINE AND [ 3 H ] D O P A
TABLE III
598 TABLE IV ['~H]DOPA
INCUBATION OF CAUDATE PARTICULATES WITH [t4C]TYROSINE
COSUBSTRATES
DOPA
AND
DOPAMINE ISOTOPE RATIOS IN THE PARTICULATES AND MEDIUM
The substrate concentrations (mlcromolar) and specific radloactJvlties (nG/nmole) respectively ~vere: [14C]tyrosme, 5.3 and 28.0; [aH]DOPA, 9.1 and 3.9. In all other respects, the incubation method was same as that described in Table III. Following the lncubahon the particulates were separated from the Incubation medium on Milhpore filters as described in the Materials and Methods. .
.
Fraction analyzed
Particulates
.
.
.
.
.
.
.
.
.
.
.
.
.
.
f 14Cf-
DOP14
DA
1.3 ± 0 1
4 5 zk 0 2
.
.
.
.
.
.
.
.
Isotope ratio (14C/3H x 100)
Productsformed (nmole/g/h)
~/14C7-
.
,73H~DA
R(DOPA)
22.6 ± 1.1
12.2 ± 0.4
R(DA)
147.0 ± 1 9 P f 0.001
Medium
9.6±04
16.8±0.7
88.0=t=5.5
13.5±0.1
1392 ~ 2.7 P (0001
DOPA and dopamine isotope ratios in the synaptosomes
The enzymes tyrosine hydroxylase and DOPA decarboxylase have been observed 4,s in the caudate nucleus synaptosomal particles. The formation o f DOPA and dopamine from the tyrosine and DOPA cosubstrates in our present experiments may occur in the particulates following the uptake of the substrates. Although studies 5,9 with synaptosomes suggest rapid uptake of exogenous amino acids and their quick equilibration with the endogenous pool, we wished to compare the isotope ratios in the particulates where the hydroxylation and decarboxylation may actually occur. For these experiments, caudate synaptosomal preparations were obtained as described in the Materials and Methods. Following their incubation with the substrate mixture of [14C]tyrosine and [SH]DOPA, the synaptosomal particulates were quickly separated from the medium by filtering on 0.8 nm Millipore filter (see Materials and Methods). Each separated fraction, particulates and medium, was then analyzed and the isotope ratios determined. The results (Table IV) indicate that the products formed were distributed between the medium and the particulates and the latter fraction contained 12%, 21% and 2 0 ~ respectively of [t4C]DOPA, [14C]dopamine and [SH]dopamine. Incidentally, there were sufficient radioactivities in these samples to allow less than 5 % counting error. The results also show that in the particulates, as well as in the medium, the R(DA) value was much larger then R(DOPA) and in fact 10.3-12.0 times larger. Our time study (Fig. 1). also reveals that the difference between the isotope ratios in the synaptosomal particles was significantly (P < 0.001 ) large during the incubation periods ranging from 10 to 45 min. DISCUSSION
The results in Table I show that both DOPA and dopamine are formed from [14C]phenylalanine and [14C]tyrosine following the incubation with the caudate
599
150
125
0
I00
m ul 0
75
N 0
~
5o
25
I0 M I N U T E S
20
30
45
p (0.001
Fig. 1. Temporal changes of intrasynaptosomal R(DOPA) and R(DA) values. For these incubations the substrate concentrations (micromolar) and specificradioactivities (nCi/nmole) respectively were: [14C]tyrosine,5.3 and 29.4; [SH]DOPA, 1.8 and 18.9. In all other respects, the experiments were as described for Table IV. preparation. Paper chromatographic separation followed by radioactivity assay carried out with a number of samples indicated only a minor contamination of dopamine by labeled norepinephrine, usually 10% or less, as we have reported 1 before. The lack of any significant norepinephrine formation permitted us to use the side chain tritium-labeled tyrosine in some of our experiments with no loss of radioactivity. We have employed relatively crude preparation of the synaptosomal-mitochondrial fraction for most of our studies. However, for some of our experiments we have prepared the fraction (P2) according to Whittaked s, washed once and incubated with [14C]tyrosine [SH]DOPA cosubstrates. The results (Table I) are essentially indistinguishable from those data from incubation of the relatively crude fractions (Table I). It may be seen from the results in Table I that the isotope ratio in dopamine formed by caudate preparations differs significantly from the ratio in DOPA. The ratio in dopamine was clearly higher than R(DOPA) from the incubation with [14C]phenylalanine in the presence of [SH]DOPA. If the [14C]DOPA product of
600 [14C]phenylalanine hydroxylatlon is freely exchangeable with the [3H]DOPA in the medium, one may rather expect a lower isotope ratio m dopamine. The [3H]DOPA present initially in the medium was being continuously decarboxylated from the very start of the incubation before any [14C]DOPA could be formed. Similarly, if the [14C]DOPA gradually formed from [l'4C]tyrosme is freely exchangeable with the [3H]DOPA present in the medium, a R(DA) value lower than R(DOPA) should result. The data (Table 1) indicate that from [14C]tyrosine [3H]DOPA cosubstrate mixture as well, R(DA) is higher than R(DOPA). In some experiments we have switched isotopes, i.e., have employed [3H]tyrosine as cosubstrate with [14C]DOPA. The results ([3H]DOPA, 4.4 ! 0.1 nmole/g/h; [3H]DA, 13.5 ± 0.1; [14C]DA, 23.3 :~0.3; R(DA), 1227 0 ~ 9.7 and R(DOPA), 134.0 ± 2.7) show again that R(DA) was much higher than R(DOPA). Since free exchange between the newly formed [14C]DOPA and exogenous [3H]DOPA does not appear to take place, some sort of compartmentation of the newly formed DOPA is indicated. The higher isotope ratio in dopamine also suggests a preferential decarboxylation of [14C]DOPA formed from either phenylalanine or tyrosine. In comparison with our findings on DOPA, the data on the oxidation of dopamine to DOPAC and the corresponding isotope ratios (Table II) do not clearly suggest any compartmentation of dopamine. When we chose [3H]DOPA as the precursor of newly formed dopamine m the presence of added [14C]dopamme, the isotope ratios in DOPAC and dopamine did not differ markedly. The isotope ratio m DOPAC was clearly lower than in dopamine from the incubation of the substrate mixture of [aH]tyrosine and [14C]dopamine. It is however possible that the exogenous dopamme and that formed in situ are not freely exchangeable and the respectlve oxidations by MAO proceeds at different rates. The reason for the compartmentation of DOPA ~s not clear at the present time but our results in Table Ill and Table IV indicate some basic facts about the compartmentatlon. Tyrosine and DOPA are expected to interact during synaptosomal uptake and subsequent enzymatic reactions. The data on the uptake of tyrosine and DOPA cosubstrates (Table IlI) show that increases of uptake resulted from the elevation of substrate concentrations. The increase of DOPA uptake from 8.3 to 79.7 nmoles/g/10 rain and that of tyrosine uptake from 9.0 to 42.3 nmoles/g/10 min (Table lII) increased the respective product formations and led to new values of R(DOPA) and R(DA). However, R(DA) values remained significantly (P < 0.001) higher (10.6-13.7 times) than that of R(DOPA) at all the substrate concentrations tested. It appears therefore that whatever may be the mechanism of the compartmentation, it is not an artifactual effect of the substrate concentrations chosen or the resultant uptakes. The results in Table IV show that the isotope ratio in the particulates, whether m DOPA or in dopamine, was very close to that in the medium. The results of analysis of the entire incubation mixture may therefore accurately reflect the values m separated synaptosomes. Since the R(DOPA) and R(DA) values from the synaptosomes differed just as significantly, the preferential decarboxylation of newly formed DOPA may occur intrasynaptosomally. To sum up, it appears that DOPA that is formed in situ by the hydroxylation of its precursors may not be freely exchangeable with exogenous DOPA because
601 o f c o m p a r t m e n t a t i o n and the newly formed D O P A m a y be preferentially decarboxylated. One o f several alternative explanations for these p h e n o m e n a is that, in the synaptosomes, the hydroxylating and the decarboxylating enzymes are organized advantageously in close proximity. A l t h o u g h such an organization has been proposed for the enzymes o f serotonin synthesis 6, our present data do not necessarily prove such an organization o f tyrosine hydroxylase and D O P A decarboxylase enzymes. Finally, the c o m p a r t m e n t a t i o n o f D O P A and that o f tyrosine as indicated by our results ~,a appear rather similar. I f indeed similar mechanisms underlie both the cases, the c o m p a r t m e n t a t i o n of tyrosine is not due to its being the p r o d u c t and substrate o f the same enzyme. Individually characteristic mechanism o f c o m p a r t m e n t a t i o n is however entirely possible. ACKNOWLEDGMENT
This work was supported by the Department of Mental Hygiene, State of New York.
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