47

Biochimica et Biophysica Acta, 523 (1978) 47--52 © Elsevier/North-Holland Biomedical Press

BBA 68371

EFFECTS OF MELANIN ON TYROSINE HYDROXYLASE AND PHENYLALANINE HYDROXYLASE

TOSHIHARU NAGATSU a, YUKIKO NUMATA(SUDO) b, TAKESHI KATO a, KENJI SUGIYAMA c and MIKI AKINO c

a Laboratory of Cell Physiology, Department of Life Chemistry, Graduate School at Nagat- suta, Tokyo Institute of Technology, Yokohama, 227, b Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Nagoya, 464, and c Department of Biology, Tokyo Metropolitan University, Tokyo, 158 (Japan)

(Received July 25th, 1977)

Summary

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demon- strated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropte- rin, depending upon its concentrations, bu t the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.

I n t r o d u c t i o n

It is well known that the essential lesion of Parkinson's disease is an idio- pathic degeneration of the nigro-striatal dopaminergic neurones which contain melanin. The concentration of dopamine in the pigmented neurones decreases simultaneously with the disappearance of melanin [1]. It has been also found that tyrosine hydroxylase activity is greatly decreased in nigro-striatum of Par- kinsonian patients [2--4]. On the other hand, Katz et al. [5] reported that various polyanions such as heparin, chondroitin sulfate and phosphatidylserine activate tyrosine hydroxylase from the bovine caudate nucleus.

Since melanin is located together with tyrosine hydroxylase in the same nigral pigmented neurones and it has polyanionic properties [6], we have exa- mined the effect of melanin on tyrosine hydroxylase from brain and adrenal

48

glands. The effect of melanin on phenylalanine hydroxylase, which is a similar tetrahydropterin~lependent monooxygenase but located in the liver cell, has been also examined for comparison.

Materials and Methods

6-Methyltetrahydropterin was purchased from Calbiochem. L-erythro-bio- pterin was isolated from the skin of the bullfrog [7], and the corresponding 5,6,7,8-tetrahydro derivative was prepared by catalytic hydrogenation in 0.1 M HC1 using platinum oxide as a catalyst [8]. Tyrosine hydroxylase from rat brain (caudate nucleus) or adrenal glands was prepared by ammonium sulfate fractionation. Rat caudate nucleus or adrenal glands was homogenized in 9 vols. of 0.32 M sucrose with a glass Potter homogenizer, and the homogenate was centrifuged at 30 000 ~g for 30 min. To the supernatant solid (NH4)2SO4 was added to give 45% saturation, and the mixture was centrifuged at 10 000 g for 20 min. The precipitate was dissolved in 20 mM potassium phosphate buffer (pH 6.2) and dialyzed against 200 vols. of 5 mM 2-(N-morpholino)- ethanesulfonic acid buffer (pH 6.2), changed 3 times. Bovine adrenal medulla tyrosine hydroxylase was purified from the soluble fraction by ammonium sulfate fractionation and column chromatography on Sephadex G-200 as described previously [9]. The enzyme solution was dialyzed against 5 mM buffer (pH 6.2) as in the case of rat brain or adrenal enzyme. Phenylalanine hy- droxylase was prepared from rat liver by the method of Sugiyama (unpublished). Dihydropteridine reductase was purified from the extract of bovine liver by the method of Hasegawa [10]. Melanin was prepared from L-dopa [6], and the col- loidal aqueous suspension was used. The concentration of melanin was tentati- vely expressed as dry weight per ml suspension.

Tyrosine hydroxylase activity was measured by estimating the formation of [14C]dopa from L-[U-14C]tyrosine [11,12]. The incubation mixture contained 0.1 M buffer (pH 6.2), 40 pg (2000 U) catalase, the enzyme, 200 pM (or at various concentrations for kinetic studies) 6-methyltetrahydropterin, 20 mM mercaptoethanol, 50 pM (or at various concentrations for kinetic studies) L-tyrosine containing 0.07 pCi L-[U-~4C]tyrosine (483 mC i / mmol ) and water to make up a total volume of 0.5 ml. Incubation was carried out in air at 30°C for 10 min. For controls water was added instead of enzyme. Boiled-enzyme controls were also carried out, and both control values were similar (about 350 cpm).

The assay mixture (1.0 ml) for phenylalanine hydroxylase contained 0.1 M potassium phosphate buffer (pH 6.8) 1 mM phenylalanine 0.2 mM NADPH 77 pg (300 mU) dihydropteridine reductase phenylalanine hydroxylase and 20 t~M L-erythro-tetrahydrobiopterin. Incubation was carried out in air at 25°C for 30 min. Tyrosine formed was determined by the nitroso-naphthol procedure [13]. Melanin did not have any effect on nitroso-naphthol assay of tyrosine. Protein was measured by the method of Lowry et al. [15].

The Km values with purified bovine adrenal tyrosine hydroxylase were deter- mined from Lineweaver-Burk plots [15] on a Facom-230 computer using Wil- kinson's program [16], and expressed as mean _+standard error of mean.

49

Results

Effect o f melanin on rat liver phenylalanine hydroxylase Melanin inhibited rat liver phenylalanine hydroxylase (Fig. 1). Preincubation

of the enzyme with melanin in the presence or absence of phenylalanine gave similar inhibition rates.

Effect o f melanin on tyrosine hydroxylase from rat brain and rat adrenal glands The dialyzed crude tyrosine hydroxylase from brain (caudate nucleus) or

adrenal glands (Fig. 2) was activated markedly by melanin; the activation of tyrosine hydroxylase was more pronounced with the brain enzyme (about 10- fold at 100 pg/ml of melanin) than that with the adrenal enzyme (about 3-fold at 100 pg of melanin). However, the activation curves with the increasing con- centration of melanin were similar between brain and adrenal enzymes. The activation was observed only in the suboptimal concentrations of tyrosine (50 pM), and 6-methyltetrahydropterin (200 pM), and not in the saturated concen- tration of tyrosine (200 pM) and 6-methyltetrahydropterin (1.5 mM). As shown in Fig. 3, rat brain enzyme showed a deviation from classical Michaelis- Menten kinetics against 6-methyltetrahydropterin in the absence of melanin and two different Km values were obtained depending on whether the concen- trations were lower or higher than 200 pM. At low concentrations (less than 200 pM) a relatively low Km value of about 120 pM was observed, whereas at higher concentrations (higher than 200 pM) the Km values was higher and registered about 2.8 mM. The melanin-activated enzyme gave a normal Michae- lis-Menten kinetics and a single Km value of about 220 pM.

2O

o ~ 4O

c

8C

I00 I = , = i

2 4 7 I0 20 Melanin. ( pcj,,'rnl )

0 ' 1 T M . . . . t . . . . t

o 0 50 I00 c~ Melanin (pg /ml )

Fig. 1. E f f e c t o f m e l a n i n on r a t l iver p h e n y l a l a n i n e h y d r o x y l a s e . T h e r e a c t i o n m i x t u r e ( to ta l v o l u m e , 1.0 ml) c o n t a i n i n g (in f inal c o n c e n t r a t i o n s ) 0.1 M p o t a s s i u m p h o s p h a t e b u f f e r (pH 6 .8) , 0.2 m M N A D P H , 300 m U (77 /zg) d i h y d r o p t c r i d i n e r e d u c t a s e , 1 m M p h e n y l a l a n i n e , 92 p g p h e n y l a l a n i n e h y d r o x y l a s e , and va r i o u s a m o u n t s o f m e l a n i n was p r e i n c u b a t e d a t 25°C fo r 10 ra in , and the r e a c t i o n w a s s t a r t e d by the a d d i t i o n o f 20 jzM t e t r a h y d r o b i o p t e r i n . T h e i n c u b a t i o n was car r ied o u t a t 25°C fo r 30 m i n .

Fig. 2. E f f e c t o f m e l a n i n on r a t b ra in ( c a u d a t e nuc leus ) or r a t ad rena l t y ro s ine h y d r o x y l a s e . The e n z y m e in the so luble f r a c t i o n was f r a c t i o n a t e d w i t h a m m o n i u m su l fa te and ex t ens ive ly d ia lyzed , as desc r ibed in the t ex t . 480 ~g o f the b r a in e n z y m e and 450 pg of the adrena l e n z y m e were used for each assay in the s t a n d a r d i n c u b a t i o n m i x t u r e i nc lud ing 50 /zM t y r o s i n e and 200 pM 6 - m e t h y l t e t r a h y d r o p t e r i n as desc r ibed in the t ex t . The va lues are the m e a n o f dup l i ca t e e x p e r i m e n t s . • e , ra t b ra in e n z y m e ; X X, ra t ad r en a l e n z y m e .

50

A

0

O.f

E

> 0

f

/ 60

, ~ " * " ' ~ a 5 0 i i

- r 0.5 1.0 1.5 2.0 / / , , , ~ .~ {6 M PH 4) (raM) 40

E 30 • 30 E

o

' ° ~ . °/° ~ •

o, 5C

0 c~O

0

o • ~ 0

5 I0 15 I i

0 I0 20 I/~MPH4) (mM) -I Time {mlnl

Fig. 3. L ineweave r -Burk a nd Michael is-Menten p lo ts i l lustrat ing the e f fec t of 6 - m e t h y l t e t r a h y d r o p t e r i n on the ra te of d o p a f o r m a t i o n by ra t b ra in ty ros ine h y d r o x y l a s e . Th e values are the m e a n of dup l ica te exper i - m e n t s . T he s t anda rd i n c u b a t i o n m i x t u r e inc luding 480 /zg of the e n z y m e , 50 pM tyros ine and 50 # g / m l me lan in was used. • • , c on t ro l (no me lan in ) ; o o, plus me l an in 50 /~g/ml.

Fig. 4. Ef fec t of m e l an in on pur i f ied bov ine adrena l ty ros ine h y d r o x y l a s e . Tyros ine h y d r o x y l a s e was puri- f ied f r o m the soluble f rac t ion of bov ine adrena l me du l l a and d ia lyzed ex tens ive ly , as descr ibed in the text . The s t anda rd i n c u b a t i o n m i x t u r e inc luding 27 /~g of the e n z y m e , 50 #M tyros ine an d 5 Izg/ml or 50 ~zg/ ml of me lan in was used. o o, con t ro l (no me la n in ) ; • o, plus me l an in 5 pg lml ; X X, plus me lan in 50 #g /ml .

----L ..L_ I--- 1,0

0.5 1.0 15 E {'6 M PH4) (m M)

J. L

0 I/{6MOpH4) (mM).120

Fig. 5. L ineweave r -Burk and Micbael is-Menten p lo ts i l lustrat ing the e f fec t of 6 - m e t b y l t e t r a h y d r o p t e r i n on the ra te of d o p a f o r m a t i o n by pur i f ied bov ine adrena l ty ros ine h y d r o x y l a s e . Th e s t anda rd i n c u b a t i o n mix- tu re including 50 /~M tyros ine and 5 # g / m l of me lan in was used. • -', con t ro l (no me lan in ) ; c, o, plus me lan in 5 /Jg/ml. The values are the m e a n of dupl ica te expe r imen t s .

51

2.0

0

/ £ . L _ _

I 0 0 20 0/, .~(Tyrosine) (.uM)

/ . L .J

0.1 0..~ V(Ty rosine] (jJM)

Fig. 6. L i new eave r -B urk and Michae l i s -Menten p lots i l lustrating the e f f e c t o f tyros ine on the ra te of dopa f o r m a t i o n by pur i f ied bovine adrenal ty ros ine h y d r o x y l a s e . The standard i n c u b a t i o n m i x t u r e including 1 .5 m M 6 - m e t h y l t e t r a h y d r o p t e r i n and 5 izg/ml of me lan in was used. ¢ ¢, contro l ; o o, plus me lan in 5 pg /ml . T he va lues are the m e a n of dupl icate e x p e r i m e n t s .

Effect of melanin on tyrosine hydroxylase purified from bovine adrenal medulla

To investigate precisely the kinetic properties of the activation of tyrosine hydroxylase by melanin, tyrosine hydroxylase was purified from bovine adrenal medulla. The purified enzyme was activated by melanin at much lower concentrations (5 pg/ml) than the crude rat adrenal or brain enzyme. Similar degrees of activation were observed either without or with preincubation of the enzyme with melanin. Melanin at higher concentrations (50 pg/ml) showed similar activation as at 5 pg/ml (Fig. 4). As shown in Fig. 5, the purified and dialyzed tyrosine hydroxylase, like crude rat brain enzyme (Fig. 3), showed a pronounced deviation from classical Michaelis-Menten kinetics against 6-methyl- tetrahydropterin in the absence of melanin; at low concentrations (less than 500 pM) of 6-methyltetrahydropterin a relatively low Km value of 102 + 15 pM was observed, whereas at higher concentrations (higher than 500 ~M) the Km value was much higher and registered 659 _+ 20 pM. In contrast, the mela- nin-activated tyrosine hydroxylase gave a single Km value of about 141 _+ 15 pM. The activation was observed only in the suboptimal concentrations of tyrosine and 6-methyltetrahydropterin. The K m value of tyrosine was measured with 6-methyltetrahydropterin cofactor at 1.5 mM. As shown in Fig. 6, a classi- cal Michaelis-Menten curve was observed. K m values of tyrosine in the absence and presence of melanin were 65 _+ 7 pM and 47 + 8 pM, respectively.

Discussion

The above data demonstrate that melanin which is present in the nigral pig- mented neurones together with tyrosine hydroxylase activates tyrosine

52

hydroxylase from brain (caudate nucleus) and adrenal glands. The stimulation by melanin occurs only in the presence of subsaturating concentrations of sub- strate and pterin cofactor. The data presented indicate that melanin affects primarily the cofactor affinity. Interestingly, another similar pterin-dependent monooxygenase, rat liver phenylalanine hydroxylase, was not activated but inhibited by melanin. The mechanism of activation of tyrosine hydroxylase by melanin may be due to the interaction of melanin with the enzyme as poly- anion, as reported by Katz et al. [5]. The polyanionic nature of melanin was described by Akino (ref. 6, see also ref. 17). The activation by melanin was observed only in the buffer with low ionic strength, and only with the exten- sively dialyzed enzyme. We reported that 6-methyltetrahydropterin showed a single Km value with the purified undialyzed bovine adrenal tyrosine hydrox- ylase in the incubation mixture with a high (0.2 M) sodium acetate buffer con- centration [9]. In the present study, it has been found that two different Km values of 6-methyltetrahydropterin were obtained with the dia lyzed enzyme and under low salt concentrations. This deviation from classical Michaelis- Menten curves was normalized by melanin as the enzyme was activated. Mela- nin may introduce a conformational change on tyrosine hydroxylase by inter- acting with the enzyme in its active site, as proposed by Katz et al. on various polyanions [ 5 ]. Although it is difficult to extrapolate present in vitro phenom- enon of melanin activation of tyrosine hydroxylase to the enzyme regula- tion in vivo, it is interesting that the natural substance which presents with tyrosine hydroxylase in the pigmented brain neurones can activate tyrosine hydroxylase.

References

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