Original Paper Ophthalmic Res 1992;24:228-233
Department of Ophthalmology, Faculty of Medicine, Kyoto University, Sakyoku, Kyoto, Japan
Keywords Tyrosine hydroxylase Bovine retina DOPA High-performance liquid chromatography Catecholamine Amacrine Cell
Simple and Accurate Assay for Tyrosine Hydroxylase from Bovine Retina by High-Performance Liquid Chromatography with Fluorescence Detection
Abstract We present a simple and sensitive assay for tyrosine hydroxy lase (TH) using high-performance liquid chromatography (HPLC) with a fluorescence detector which enabled us to study bovine retinal TH of a small quantity in crude retinal samples. The detection limit with this method was 1 pmol of dihydroxyphenylalanine (DOPA) enzymatically produced. Retinal TH exhibited the maximum activity at pH 6 to 6.3, and almost none at physiological pH of above 7, a tendency similar to that reported for striatal TH.
Introduction Tyrosine hydroxylase (TH) is the first and the rate-limiting enzyme in catecholamine biosynthesis [1,2]. The enzyme catalyzes the conversion of tyrosine to dihydroxyphenylal anine (DOPA) and requires molecular oxygen and the pteridine cofactor. For the assay of TH, radioisotopic methods have been wide ly used [3, 4], Recently with the introduction of high-performance liquid chromatography
Received: November 11. 1991 Accepted: March 9, 1992
(HPLC), TH assay became more handy and no less sensitive than the assay with radioiso topes [5-8]. Still it often required complicated procedures and/or skillful techniques. We as sayed TH in bovine retina without any pre treatment by measuring the native fluores cence of DOPA in combination with HPLC using a prevalent and reliable octadecylsilane (ODS) column. Some properties of bovine retinal TH assayed by the present method are also described.
Masayoshi Iwaki. MD Department of Ophthalmology Kyoto University Faculty of Medicine Kyoto 606 (Japan)
© 1992 S. Karger AG. Basel 0030-3747/92/ 0244-0228S2.75/0
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
Michiko Mandai Masayoshi Iwaki Yoshihito Honda
Materials /.-Tyrosine, catalase, o-benzylhydroxylamine (an aromatic L-amino acid decarboxylase inhibitor), £>/.-6-methyl-5,6,7,8-tctrahydropterinc(6MPH4), dihydropteridine reductase and NADH were pur chased from Sigma. L-DOPA was obtained from Wako Pure Chemicals. (6R.S)-5.6,7,8-tctrahydro-Z.biopterin (BH4) was from Dr. B. Schircks Laborato ries, Jona, Switzerland. All other chemicals used were of analytical grade. Enzyme Preparation Bovine eyes were purchased from a local slaughter house and kept in ice for a couple of hours until retina was detached in the laboratory. The retina (5-10 g, wet weight) was immediately homogenized with a teflon homogenizer in one volume (w/v) of Dulbecco’s phos phate buffer saline (PBS) containing protease inhibi tors (4 pg/ml of p-amidinophenylmethanesulfonylfluoride and 10pg/ml of leupeptin) and centrifuged at 16.000 rpm for 60 min. The supernatant was passed through a Sephadex G-25 column to remove low mo lecular weight substances. The final preparation (ap prox. 15-20 mg protein/ml) was stored at -8 0 °C until use. All the procedures described were performed be low 4°C in the shortest period. Bovine striatum and adrenal medulla were prepared accordingly. Assay o f T il The standard assay method was as follows: retinal homogenates (0.2-2 mg protein) were incubated with 0.05 mM tyrosine, 1 mM BH4 (freshly prepared before each experiment), 3.5 m.V/NADH, 0.02 units of dihydroxyptcridinc reductase (1 unit oxidizes 1.0 pmol NADH to NAD per minute), 15 pg catalase, 40 mM sodium acetate buffer (pH 6.0), 0.1 m l/ o-benzylhydroxylamine in the total volume of 100 pi. The mix ture was incubated at 37 °C for 5-20 min and the reac tion was stopped by adding 100 pi of 0.5 M perchloric acid containing 0.4 mM sodium mctabisulphite and 0.1 m.W disodium ethylcncdiaminetetraacetatc (diso dium EDTA). The mixture was centrifuged at 16.000 rpm for 20 min and the supernatant was di rectly injected into HPLC. HPLC Analysis HPLC assay was performed according to the modi fied method of Naoi et al. [8], The HPLC system (Shimadzu LC-6A) equipped with an ODS column (TSKODS-120T, 4.6 mm X 25 cm), a fluorescence detector (RF-535) and a data processor (C-R6A) was used. A
guard column (TSK Guardgcl ODS-120T) was placed in front of the separation column. Mobile phase was 90 m l/ sodium acetate, 35 m l/ citric acid, 0.13 mM disodium EDTA, 0.23 m.W sodium n-octanesulphonate with 10.5% methanol. The flow rate was 1 ml/min. The excitation and emission wavelengths were 281 and 314 nm. respectively. The enzyme activity was expressed as pmol DOPA produced per minute. For the standard, appropriate amount of authentic DOPA was used.
Results As shown in figure 1. a linear relationship was obtained between the amount of pure DOPA injected into HPLC (5-100 pmol) and the fluorescence intensity presented as inte grated peak area. We could detect as low as 1 pmol DOPA with the base line noise mini mized almost to the negligible level. The re producibility of the chromatogram was reli able (error within 5%). The stock solution of DOPA standard (2 mM in 5 mM HC1) was stable on chromatogram at least for 2 weeks. The results of the TH assay with crude reti nal samples are shown in figure 2. The peak corresponding to DOPA was sharply sepa rated from other significant peaks and the retention time of DOPA was approximately 3.9 min and that of tyrosine 4.8 min. The mixture with the boiled enzyme or without BH4, where there should be no enzymatic DOPA formation, resulted in no detectable amount of DOPA by our analysis system. This indicates that there was no significant endogenous factor interfering the fluores cence of enzymatically formed DOPA after the assay procedure. The reaction was timedependent up to 30 min of incubation and the velocity of DOPA production increased lin early in accordance with the amount of the enzyme added (data not shown). An addition of o-benzylhydroxylamine, an inhibitor of DOPA decarboxylase, made no difference in 229
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Materials and Methods
2 O
5
Fig. 1. Standard analysis of DOPA by HPLC. A Recording of HPLC analysis. 1: lOOpmol; 2: 50 pmol: 3: 20 pmol; 4: 10 pmol; 5: 5 pmol of DOPA injected. B Cor relation between the amount of DOPA injected and integrated peak area representing fluores cence intensity.
-
Of—.---■
---------------- r - T —
B
0
.-------- t —
50 pmol/injection
.---------------- 1
100
Lr: A
1'
Fig. 2. HPLC analysis of the reaction products, a: DOPA (r.t. = 3.9 min); b: tyrosine (r.t. = 4.8 min). A Complete assay mixture. B Assay without BH4. C Assay with boiled enzyme.
B
R
DOPA production at any pH in our assay sys tem. The assay was performed in triplicate and the error was within 10%. We examined some enzymological proper ties of bovine retinal TH using our assay method. The optimal pH was revealed to be 6.0 with sodium acetate buffer and 6.3 with 2-(N-morpholino) ethanesulfonic acid buffer
230
Mandai/Iwaki/Honda
---------------------------------------- b
(MES). both of which gave almost the same level of maximum specific activity of approxi mately 40 pmol/min/mg protein (fig. 3). TH activity at pH above 7 was less than 20% of the maximum activity. Specific activities of TH in crude extracts of bovine striatum and adrenal medulla were 6 nmol/min/mg protein and 10 nmol/min/mg protein, respectively.
Assay for Retinal Tyrosine Hydroxylase
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
'
Fig. 3. pH profile of tyrosine hydroxylase in the crude extracts of bovine retina. sodium acetate buffer; -A-: MES buffer; -o-: HEPES buffer.
Fig. 4. Effect of tyrosine concentration on tyrosine hydroxylase reaction velocity. The reaction was per formed according to the standard assay method.
The results of this kinetical study for retinal TH are summarized in table 1; Kmfor the nat ural cofactor BH4 was 270 1xM and that of 6MPH4, a synthetic pteridine cofactor, was 310 pAf; the maximum velocity was higher for the natural cofactor. For tyrosine, Kmwas 6.7 \iM and substrate inhibition was observed when tyrosine concentration was above 0.05 mM as shown in figure 4.
Table 1. Kinetical parameters of bovine retinal tyrosonc hydroxylase
Tyrosine bh4 6MPH4
Km, flAf
Vmax, Pmol/ min/mg
6.7 270 310
42.5 42.0 32.0
The assay was performed according to the standard assay method (pH 6.0).
There have been several TH assay methods using HPLC published so far [5-8], A good sensitivity was obtained with these methods but some required time-consuming pretreat ment with aluminum oxide [5-7] or some particular maintenance of electrochemical de tector [5, 6, 8], Our aim was to avoid such complicated procedures and to use an ODS
column and fluorescence detector, which are both prevalent and considered to be reliable. For the ODS chromatography we chose the method of Naoi et al. [8] with a modification to use a fluorescence detector. In our method, a good correlation was obtained between the pmol range of DOPA injected into HPLC and the fluorescence intensity. This high sensitiv
231
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Discussion
ity was well retained in actual analysis of crude extracts after the described assay proce dure (fig. 1,2). This was almost a comparable sensitivity to those of previously reported as say methods [3-8]. Because of the minimized procedure before HPLC analysis (addition of perchloric acid and centrifugation), our method was considered to be accurate enough without any correction using an internal stan dard. In order to confirm the reliability of our method, we simultaneously assayed TH in bovine striatum and adrenal medulla and ob tained values (approx. 6 and 10 nmol/min/mg protein, respectively) similar to the results previously published for their specific activi ties [9, 10], For the protection of the separa tion column we used a small guard column and could reproduce stable results for at least up to 1,000 assays per guard column. As another advantage of our method, kinetical studies of retinal TH with different concen trations of substrate were easily handled. We suggest this practical and sensitive method is applicable to any tissue, especially with low TH activity like retina. In our enzymological study of retinal TH, the activity seemed to be suppressed at physi ological pH range of around 7.2. Okuno et al. [ 10, 11 ] reported that crude striatal TH was in more suppressed state than crude adrenal TH
at physiological pH. They suggested it to be due to the difference of inhibitory condition by end products in the crude extracts [11, 12], According to their speculation, retinal TH may be in a similarly suppressed state as stria tal TH. For this difference in activity at physi ological pH, however, further investigations are required to pursue some other possibili ties, as for instance difference of molecular species in different organs. Several mechanisms have been reported to regulate TH including phosphorylation by various protein kinases [13, 14], In vertebrate retina, recent immunohistochcmical studies revealed that TH was localized in dopaminer gic amacrine cells [15, 16], and it has been reported to be activated by light exposure [ 17], The detailed mechanism of activation of retinal TH by light exposure or other possible stimulation has not been solved. We believe that the simple method we presented here will help to study regulatory properties and roles of retinal TH.
Acknowledgment This study was partly supported by the Grant-inaid for Scientific Research 02454402 from the Minis try of Education, Science and Culture of the Japanese Government.
1 Nagatsu T. Levitt M, Udenfriend S: Tyrosine hydroxylase: The initial step in norepinephrine biosynthesis. J Biol Chem 1964:239:2910-2917. 2 Brennenman AR. Kaufman S: The role of tetrahydropteridines in the enzymatic conversion of tyrosine to 3.4-dihydroxyphenylalanine. Biochem Biophys Res Commun 1964: 17:177-183.’
232
3 Nagatsu T, Levitt M, Udenfriend S: A rapid and simple radioassav for tyrosine hydroxylase activity. Anal Biochem 1964;9:122-126. 4 Okuno S. Fujisawa H: Assay of tyro sine 3-monooxygenase using the coupled nonenzymatic decarboxyla tion of Dopa. Anal Biochem 1983; 129:405-411.
Mandai/lwaki/Honda
5 Nagatsu T, Oka K, Kato T: Highly sensitive assay for tyrosine hydroxy lase activity by high performance liquid chromatography. J Chromatogr 1979;163:247-252. 6 Philipp E: Assay for tyrosine hy droxylase in hypothalamic homoge nates using high-performance liquid chromatography with electrochemi cal detection. J Chromatogr 1987; 419:27-36.
Assay for Retinal Tyrosine Hydroxylase
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
References
11 Okuno S, Fujisawa H: The activity of tyrosine 3-monooxygenase is usually suppressed by an endoge nous factor in brain. Biochem Biophys Res Commun 1984; 124:223— 228. 12 Okuno S. Fujisawa H: A new mech anism for regulation o f tyrosine 3monooxygenase by end product and cyclic AMP-dependent protein ki nase. J Biol Chem 1985:260:26332635. 13 Campbell DG, Hardie G, Vulliet PR: Identification of four phosphor ylation sites in the N-terminal re gion of tyrosine hydroxylase. J Biol Chem 1986:261:10489-10492. 14 Griffith LC, Schulman H: The mul tifunctional Ca27calmodulin de pendent protein kinase mediates Ca-'-dependent phosphorylation of tyrosine hydroxylase. J Biol Chem 1988;263:9542-9549.
15 Mitrofanis J, Maslim J. Stone J: On togeny of catecholaminergic and cholinergic cell distributions in the cat’s retina. J Comp Neurol 1989; 289:228-246. 16 Zhu B. Straznicky C: Dendritic mor phology and retinal distribution of tyrosine hydroxylase-like immunoreactive amacrine cells in Bufo marinus. Anat Embryol 1990:181:365371. 17 Iuvone PM, Galli CL, GarrisonGund CK, Neff NH: Light stimu lates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons. Science 1978; 202:901-902.
233
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
7 Lee M, Nohta H, Umegae Y, Ohkura Y: Assay for tyrosine hydroxy lase by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1987;415: 289-296. 8 Naoi M, Takahashi Y, Nagatsu T: Simple assay procedure for tyrosine hydroxylase activity by high-perfor mance liquid chromatography em ploying coulometric detection with minimal sample preparation. J Chromatogr 1988;427:229-238. 9 Okuno S, Fujisawa H: Purification and some properties of tyrosine 3monooxygenase from rat adrenal. Eur J Biochem 1982;122:49-55. 10 Okuno S. Fujisawa H: A compara tive study of tyrosine 3-monooxygenase from rat adrenal and brain stem. J Biochem 1985;97:265-273.
Department of Ophthalmology, Faculty of Medicine, Kyoto University, Sakyoku, Kyoto, Japan
Keywords Tyrosine hydroxylase Bovine retina DOPA High-performance liquid chromatography Catecholamine Amacrine Cell
Simple and Accurate Assay for Tyrosine Hydroxylase from Bovine Retina by High-Performance Liquid Chromatography with Fluorescence Detection
Abstract We present a simple and sensitive assay for tyrosine hydroxy lase (TH) using high-performance liquid chromatography (HPLC) with a fluorescence detector which enabled us to study bovine retinal TH of a small quantity in crude retinal samples. The detection limit with this method was 1 pmol of dihydroxyphenylalanine (DOPA) enzymatically produced. Retinal TH exhibited the maximum activity at pH 6 to 6.3, and almost none at physiological pH of above 7, a tendency similar to that reported for striatal TH.
Introduction Tyrosine hydroxylase (TH) is the first and the rate-limiting enzyme in catecholamine biosynthesis [1,2]. The enzyme catalyzes the conversion of tyrosine to dihydroxyphenylal anine (DOPA) and requires molecular oxygen and the pteridine cofactor. For the assay of TH, radioisotopic methods have been wide ly used [3, 4], Recently with the introduction of high-performance liquid chromatography
Received: November 11. 1991 Accepted: March 9, 1992
(HPLC), TH assay became more handy and no less sensitive than the assay with radioiso topes [5-8]. Still it often required complicated procedures and/or skillful techniques. We as sayed TH in bovine retina without any pre treatment by measuring the native fluores cence of DOPA in combination with HPLC using a prevalent and reliable octadecylsilane (ODS) column. Some properties of bovine retinal TH assayed by the present method are also described.
Masayoshi Iwaki. MD Department of Ophthalmology Kyoto University Faculty of Medicine Kyoto 606 (Japan)
© 1992 S. Karger AG. Basel 0030-3747/92/ 0244-0228S2.75/0
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
Michiko Mandai Masayoshi Iwaki Yoshihito Honda
Materials /.-Tyrosine, catalase, o-benzylhydroxylamine (an aromatic L-amino acid decarboxylase inhibitor), £>/.-6-methyl-5,6,7,8-tctrahydropterinc(6MPH4), dihydropteridine reductase and NADH were pur chased from Sigma. L-DOPA was obtained from Wako Pure Chemicals. (6R.S)-5.6,7,8-tctrahydro-Z.biopterin (BH4) was from Dr. B. Schircks Laborato ries, Jona, Switzerland. All other chemicals used were of analytical grade. Enzyme Preparation Bovine eyes were purchased from a local slaughter house and kept in ice for a couple of hours until retina was detached in the laboratory. The retina (5-10 g, wet weight) was immediately homogenized with a teflon homogenizer in one volume (w/v) of Dulbecco’s phos phate buffer saline (PBS) containing protease inhibi tors (4 pg/ml of p-amidinophenylmethanesulfonylfluoride and 10pg/ml of leupeptin) and centrifuged at 16.000 rpm for 60 min. The supernatant was passed through a Sephadex G-25 column to remove low mo lecular weight substances. The final preparation (ap prox. 15-20 mg protein/ml) was stored at -8 0 °C until use. All the procedures described were performed be low 4°C in the shortest period. Bovine striatum and adrenal medulla were prepared accordingly. Assay o f T il The standard assay method was as follows: retinal homogenates (0.2-2 mg protein) were incubated with 0.05 mM tyrosine, 1 mM BH4 (freshly prepared before each experiment), 3.5 m.V/NADH, 0.02 units of dihydroxyptcridinc reductase (1 unit oxidizes 1.0 pmol NADH to NAD per minute), 15 pg catalase, 40 mM sodium acetate buffer (pH 6.0), 0.1 m l/ o-benzylhydroxylamine in the total volume of 100 pi. The mix ture was incubated at 37 °C for 5-20 min and the reac tion was stopped by adding 100 pi of 0.5 M perchloric acid containing 0.4 mM sodium mctabisulphite and 0.1 m.W disodium ethylcncdiaminetetraacetatc (diso dium EDTA). The mixture was centrifuged at 16.000 rpm for 20 min and the supernatant was di rectly injected into HPLC. HPLC Analysis HPLC assay was performed according to the modi fied method of Naoi et al. [8], The HPLC system (Shimadzu LC-6A) equipped with an ODS column (TSKODS-120T, 4.6 mm X 25 cm), a fluorescence detector (RF-535) and a data processor (C-R6A) was used. A
guard column (TSK Guardgcl ODS-120T) was placed in front of the separation column. Mobile phase was 90 m l/ sodium acetate, 35 m l/ citric acid, 0.13 mM disodium EDTA, 0.23 m.W sodium n-octanesulphonate with 10.5% methanol. The flow rate was 1 ml/min. The excitation and emission wavelengths were 281 and 314 nm. respectively. The enzyme activity was expressed as pmol DOPA produced per minute. For the standard, appropriate amount of authentic DOPA was used.
Results As shown in figure 1. a linear relationship was obtained between the amount of pure DOPA injected into HPLC (5-100 pmol) and the fluorescence intensity presented as inte grated peak area. We could detect as low as 1 pmol DOPA with the base line noise mini mized almost to the negligible level. The re producibility of the chromatogram was reli able (error within 5%). The stock solution of DOPA standard (2 mM in 5 mM HC1) was stable on chromatogram at least for 2 weeks. The results of the TH assay with crude reti nal samples are shown in figure 2. The peak corresponding to DOPA was sharply sepa rated from other significant peaks and the retention time of DOPA was approximately 3.9 min and that of tyrosine 4.8 min. The mixture with the boiled enzyme or without BH4, where there should be no enzymatic DOPA formation, resulted in no detectable amount of DOPA by our analysis system. This indicates that there was no significant endogenous factor interfering the fluores cence of enzymatically formed DOPA after the assay procedure. The reaction was timedependent up to 30 min of incubation and the velocity of DOPA production increased lin early in accordance with the amount of the enzyme added (data not shown). An addition of o-benzylhydroxylamine, an inhibitor of DOPA decarboxylase, made no difference in 229
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
Materials and Methods
2 O
5
Fig. 1. Standard analysis of DOPA by HPLC. A Recording of HPLC analysis. 1: lOOpmol; 2: 50 pmol: 3: 20 pmol; 4: 10 pmol; 5: 5 pmol of DOPA injected. B Cor relation between the amount of DOPA injected and integrated peak area representing fluores cence intensity.
-
Of—.---■
---------------- r - T —
B
0
.-------- t —
50 pmol/injection
.---------------- 1
100
Lr: A
1'
Fig. 2. HPLC analysis of the reaction products, a: DOPA (r.t. = 3.9 min); b: tyrosine (r.t. = 4.8 min). A Complete assay mixture. B Assay without BH4. C Assay with boiled enzyme.
B
R
DOPA production at any pH in our assay sys tem. The assay was performed in triplicate and the error was within 10%. We examined some enzymological proper ties of bovine retinal TH using our assay method. The optimal pH was revealed to be 6.0 with sodium acetate buffer and 6.3 with 2-(N-morpholino) ethanesulfonic acid buffer
230
Mandai/Iwaki/Honda
---------------------------------------- b
(MES). both of which gave almost the same level of maximum specific activity of approxi mately 40 pmol/min/mg protein (fig. 3). TH activity at pH above 7 was less than 20% of the maximum activity. Specific activities of TH in crude extracts of bovine striatum and adrenal medulla were 6 nmol/min/mg protein and 10 nmol/min/mg protein, respectively.
Assay for Retinal Tyrosine Hydroxylase
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
'
Fig. 3. pH profile of tyrosine hydroxylase in the crude extracts of bovine retina. sodium acetate buffer; -A-: MES buffer; -o-: HEPES buffer.
Fig. 4. Effect of tyrosine concentration on tyrosine hydroxylase reaction velocity. The reaction was per formed according to the standard assay method.
The results of this kinetical study for retinal TH are summarized in table 1; Kmfor the nat ural cofactor BH4 was 270 1xM and that of 6MPH4, a synthetic pteridine cofactor, was 310 pAf; the maximum velocity was higher for the natural cofactor. For tyrosine, Kmwas 6.7 \iM and substrate inhibition was observed when tyrosine concentration was above 0.05 mM as shown in figure 4.
Table 1. Kinetical parameters of bovine retinal tyrosonc hydroxylase
Tyrosine bh4 6MPH4
Km, flAf
Vmax, Pmol/ min/mg
6.7 270 310
42.5 42.0 32.0
The assay was performed according to the standard assay method (pH 6.0).
There have been several TH assay methods using HPLC published so far [5-8], A good sensitivity was obtained with these methods but some required time-consuming pretreat ment with aluminum oxide [5-7] or some particular maintenance of electrochemical de tector [5, 6, 8], Our aim was to avoid such complicated procedures and to use an ODS
column and fluorescence detector, which are both prevalent and considered to be reliable. For the ODS chromatography we chose the method of Naoi et al. [8] with a modification to use a fluorescence detector. In our method, a good correlation was obtained between the pmol range of DOPA injected into HPLC and the fluorescence intensity. This high sensitiv
231
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
Discussion
ity was well retained in actual analysis of crude extracts after the described assay proce dure (fig. 1,2). This was almost a comparable sensitivity to those of previously reported as say methods [3-8]. Because of the minimized procedure before HPLC analysis (addition of perchloric acid and centrifugation), our method was considered to be accurate enough without any correction using an internal stan dard. In order to confirm the reliability of our method, we simultaneously assayed TH in bovine striatum and adrenal medulla and ob tained values (approx. 6 and 10 nmol/min/mg protein, respectively) similar to the results previously published for their specific activi ties [9, 10], For the protection of the separa tion column we used a small guard column and could reproduce stable results for at least up to 1,000 assays per guard column. As another advantage of our method, kinetical studies of retinal TH with different concen trations of substrate were easily handled. We suggest this practical and sensitive method is applicable to any tissue, especially with low TH activity like retina. In our enzymological study of retinal TH, the activity seemed to be suppressed at physi ological pH range of around 7.2. Okuno et al. [ 10, 11 ] reported that crude striatal TH was in more suppressed state than crude adrenal TH
at physiological pH. They suggested it to be due to the difference of inhibitory condition by end products in the crude extracts [11, 12], According to their speculation, retinal TH may be in a similarly suppressed state as stria tal TH. For this difference in activity at physi ological pH, however, further investigations are required to pursue some other possibili ties, as for instance difference of molecular species in different organs. Several mechanisms have been reported to regulate TH including phosphorylation by various protein kinases [13, 14], In vertebrate retina, recent immunohistochcmical studies revealed that TH was localized in dopaminer gic amacrine cells [15, 16], and it has been reported to be activated by light exposure [ 17], The detailed mechanism of activation of retinal TH by light exposure or other possible stimulation has not been solved. We believe that the simple method we presented here will help to study regulatory properties and roles of retinal TH.
Acknowledgment This study was partly supported by the Grant-inaid for Scientific Research 02454402 from the Minis try of Education, Science and Culture of the Japanese Government.
1 Nagatsu T. Levitt M, Udenfriend S: Tyrosine hydroxylase: The initial step in norepinephrine biosynthesis. J Biol Chem 1964:239:2910-2917. 2 Brennenman AR. Kaufman S: The role of tetrahydropteridines in the enzymatic conversion of tyrosine to 3.4-dihydroxyphenylalanine. Biochem Biophys Res Commun 1964: 17:177-183.’
232
3 Nagatsu T, Levitt M, Udenfriend S: A rapid and simple radioassav for tyrosine hydroxylase activity. Anal Biochem 1964;9:122-126. 4 Okuno S. Fujisawa H: Assay of tyro sine 3-monooxygenase using the coupled nonenzymatic decarboxyla tion of Dopa. Anal Biochem 1983; 129:405-411.
Mandai/lwaki/Honda
5 Nagatsu T, Oka K, Kato T: Highly sensitive assay for tyrosine hydroxy lase activity by high performance liquid chromatography. J Chromatogr 1979;163:247-252. 6 Philipp E: Assay for tyrosine hy droxylase in hypothalamic homoge nates using high-performance liquid chromatography with electrochemi cal detection. J Chromatogr 1987; 419:27-36.
Assay for Retinal Tyrosine Hydroxylase
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/6/2019 2:06:10 PM
References
11 Okuno S, Fujisawa H: The activity of tyrosine 3-monooxygenase is usually suppressed by an endoge nous factor in brain. Biochem Biophys Res Commun 1984; 124:223— 228. 12 Okuno S. Fujisawa H: A new mech anism for regulation o f tyrosine 3monooxygenase by end product and cyclic AMP-dependent protein ki nase. J Biol Chem 1985:260:26332635. 13 Campbell DG, Hardie G, Vulliet PR: Identification of four phosphor ylation sites in the N-terminal re gion of tyrosine hydroxylase. J Biol Chem 1986:261:10489-10492. 14 Griffith LC, Schulman H: The mul tifunctional Ca27calmodulin de pendent protein kinase mediates Ca-'-dependent phosphorylation of tyrosine hydroxylase. J Biol Chem 1988;263:9542-9549.
15 Mitrofanis J, Maslim J. Stone J: On togeny of catecholaminergic and cholinergic cell distributions in the cat’s retina. J Comp Neurol 1989; 289:228-246. 16 Zhu B. Straznicky C: Dendritic mor phology and retinal distribution of tyrosine hydroxylase-like immunoreactive amacrine cells in Bufo marinus. Anat Embryol 1990:181:365371. 17 Iuvone PM, Galli CL, GarrisonGund CK, Neff NH: Light stimu lates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons. Science 1978; 202:901-902.
233
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7 Lee M, Nohta H, Umegae Y, Ohkura Y: Assay for tyrosine hydroxy lase by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1987;415: 289-296. 8 Naoi M, Takahashi Y, Nagatsu T: Simple assay procedure for tyrosine hydroxylase activity by high-perfor mance liquid chromatography em ploying coulometric detection with minimal sample preparation. J Chromatogr 1988;427:229-238. 9 Okuno S, Fujisawa H: Purification and some properties of tyrosine 3monooxygenase from rat adrenal. Eur J Biochem 1982;122:49-55. 10 Okuno S. Fujisawa H: A compara tive study of tyrosine 3-monooxygenase from rat adrenal and brain stem. J Biochem 1985;97:265-273.
