Journul of Neuruchemistry, 1977. Vol. 29, pp. 743-746. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION Radiometric assay of tyrosine hydroxylase and tryptophan hydroxylase by Kalignost extraction procedures (Received 26 April 1977. Accepted 2 M a y 1977)
SENSITIVE radiometric assays in current use for tyrosine fractionation (WAYMIREet al., 1971) and assayed as dehydroxylase (EC 1.14.16.2)measure the radioactive DOPA scribed below, after dialysis overnight (4°C) against 10 mM(COYLE,1972; BLACK,1975) or water (SEGAL & KUCZENSKI, sodium phosphate (pH 7.0) containing 20% glycerol. 1974) produced from labelled tyrosine. Tryptophan 5-hyd- Samples were stored at -70°C. Locus coerujeus or adrenal pairs, samples of corpus striaroxylase (EC 1.14.16.4) assays measure the labelled melatonin derived from 5-hydroxytryptophan (KIZERet al., tum, brain stem, frontal cortex or cerebellum (male Wistar 1975). Dopamine and 5-HT, however, the end-products of rats. 150-200 g), were homogenised for 1 min in 9 vol of hydroxylations in the presence of aromatic L-amino acid 0.32~-sucrose at 0°C (5000 rev./min in a glass-Teflon decarboxylase (ICHIYAMA et al., 1970; WAYMIRE, 1971), are homogeniser). Triton X-100 was then added to give a final more basic than their precursors. Thus assays are sug- concentration in the homogenate of 0.5% (v/v). gested in which labelled dopamine or 5-HT is recovered Tyrosine hydroxylase reaction mixtures contained (final by extraction into 3-heptanone, as complexes formed by concentrations): 0.2 M-sodium acetate, pH 6.0; 0.5 mM-ferreaction with sodium tetraphenylboron (Kalignost). A pro- rous ammonium sulphate; 10 mwdithiothreitol; 2 mMcedure of this kind was devised for assaying choline acetyl- DMPH4; 10 pM-pyridoxd phosphate; 0.16 mM-pargyline; transferase (EC 2.3.1.6) by separation of [14C]acetylcholine 2 pl-decarboxylase preparation (assayed as described from [14C]acetyl-CoA (FONNUM, 1969). Similar procedures below: V,,, = 288nmol. dopamine.h-'., apparent K, = were subsequently used to assay the decarboxylation of 750 p ~ and ) 0.6-100p~-~-[~H]tyrosine(1.7 x lo6 d.p.m.), DOPA (BROCH& FONNUM, 1972; EMSONe f a!., 1974) and in a final volume of 20 pl. Labelled tyrosine sufficient for 5-hydroxytryptophan (EMSON& FONNUM, 1974). As a sim- 1 day's assays was freeze dried immediately before use. plification of the assay for choline acetyltransferase, the Incubation times were up to 30min (see text). scintillation mixture was used as the extraction solvent Tryptophan hydroxylase was assayed under similar conditions using 2 ~ M - ~ - M P H instead , of DMPH4 (TONG& (FONNUM, 1975). We describe here the application of procedures of this KAUFMAN,1975), 4-15 p~-~-[~H]tryptophan (1.17 x lo6 kind to the assay of tyrosine hydroxylase. The assays are d.p.m.) or 1 3 4 0 p ~ - ~ - [ ' ~ c ] t r y p t o p h a(9.2 n x lo4 d.p.m.) comparable in sensitivity with those described previously, and 4 pl-decarboxylase preparation. Incubations were for but they facilitate the management of large numbers of up to 60 min (see text). samples. Assays using Kalignost extractions are compared The decarboxylase was assayed under the conditions of for tyrosine hydroxylase, tryptophan hydroxylase, and aro- the tyrosine hydroxylase reactions using 1.0 to 800 p ~ matic L-amino acid decarboxylase. L-[~H]DOPA (1.7 x lo6 d.p.m.) and unlabelled tyrosine (final concentrations, G100 p ~ as , required). Alternatively, OXY (1.06 x lo6 5.M.O ~ M - D , L - ~ - ~ Y ~ T [3H]tryptophan MATERIALS AND METHODS d.p.m.) and 14.4 pM-unlabelled L-tryptophan were used for C3H]Water; ~-[3,5-~H]tyrosine; ~-[G-~H]tryptophan; assays under the conditions of the tryptophan hydroxylase ~-3,4-dihydroxy[ring-2,5,6-~H]phenylalaninereactions. Incubations were for 5 and 15 min, respectively. (C3H]D0PA); [3H]dopamine hydrochloride; ~ , ~ - 5 - h y d - All labelled substrates were freeze dried immediately before r~xy[G-~H]tryptophanand ~-[methylene-'~C]tryptophan use. The reactions were at 37°C in small glass tubes were obtained from the Radiochemical Centre, Amersham, Bucks. 6,7-Dimethyl-5,6,7,8-tetrahydropterinehydrochlor- (10 x 5 mm). To 10 pl samples of tissue homogenates were ide (DMPH,) and ~,~-6-methyl-5,6,7,8-tetrahydropterine added 10 p1 of the substrate mixture lacking only the hydroxylase. After incubation, the small tubes were placed (6-MPH4) were obtained from Calbiochem Ltd., and stored under nitrogen ( - 20°C). Activated alumina (acidic) in scintillation vials and the contents were washed out with 5ml of an ice cold solution of dopamine, or 5-HT from BDH Chemicals Ltd., was used without further treatment. All other reagents were of the highest purity avail- (0.25 mM), and ascorbic acid ( I mM) in 10 mM-sodium phosable. Solutions of DMPH4 or 6-MPH4, ferrous ammonium phate, pH 7.0. Acetonitrile (2 ml) containing 30 mg of sulphate, pargyline, dithiothreitol, dopamine, 5-HT and sodium tetraphenylboron was thoroughly mixed with the ascorbic acid were prepared immediately before use. resulting solution and 10 ml of scintillant (FONNUM, 1975) [3H]Tyrosine was purified before use by chroma- was added. After gentle shaking for 1 min, the vials were placed in a scintillation counter, allowed to stand for a tography on alumina and on Dowex and stored (-20°C) in absolute ethanol (COYLE,1972). minimum of 10min and counted (efficiencies: 3H, 24%; Aromatic L-amino acid decarboxylase was extracted I4C, 90%) within 12 h. This avoids quenching by coloured from pig kidneys, partially purified by ammonium sulphate oxidation products that appear after approx 24 h. 743
"
SHORT COMMUNICATION Radiometric assay of tyrosine hydroxylase and tryptophan hydroxylase by Kalignost extraction procedures (Received 26 April 1977. Accepted 2 M a y 1977)
SENSITIVE radiometric assays in current use for tyrosine fractionation (WAYMIREet al., 1971) and assayed as dehydroxylase (EC 1.14.16.2)measure the radioactive DOPA scribed below, after dialysis overnight (4°C) against 10 mM(COYLE,1972; BLACK,1975) or water (SEGAL & KUCZENSKI, sodium phosphate (pH 7.0) containing 20% glycerol. 1974) produced from labelled tyrosine. Tryptophan 5-hyd- Samples were stored at -70°C. Locus coerujeus or adrenal pairs, samples of corpus striaroxylase (EC 1.14.16.4) assays measure the labelled melatonin derived from 5-hydroxytryptophan (KIZERet al., tum, brain stem, frontal cortex or cerebellum (male Wistar 1975). Dopamine and 5-HT, however, the end-products of rats. 150-200 g), were homogenised for 1 min in 9 vol of hydroxylations in the presence of aromatic L-amino acid 0.32~-sucrose at 0°C (5000 rev./min in a glass-Teflon decarboxylase (ICHIYAMA et al., 1970; WAYMIRE, 1971), are homogeniser). Triton X-100 was then added to give a final more basic than their precursors. Thus assays are sug- concentration in the homogenate of 0.5% (v/v). gested in which labelled dopamine or 5-HT is recovered Tyrosine hydroxylase reaction mixtures contained (final by extraction into 3-heptanone, as complexes formed by concentrations): 0.2 M-sodium acetate, pH 6.0; 0.5 mM-ferreaction with sodium tetraphenylboron (Kalignost). A pro- rous ammonium sulphate; 10 mwdithiothreitol; 2 mMcedure of this kind was devised for assaying choline acetyl- DMPH4; 10 pM-pyridoxd phosphate; 0.16 mM-pargyline; transferase (EC 2.3.1.6) by separation of [14C]acetylcholine 2 pl-decarboxylase preparation (assayed as described from [14C]acetyl-CoA (FONNUM, 1969). Similar procedures below: V,,, = 288nmol. dopamine.h-'., apparent K, = were subsequently used to assay the decarboxylation of 750 p ~ and ) 0.6-100p~-~-[~H]tyrosine(1.7 x lo6 d.p.m.), DOPA (BROCH& FONNUM, 1972; EMSONe f a!., 1974) and in a final volume of 20 pl. Labelled tyrosine sufficient for 5-hydroxytryptophan (EMSON& FONNUM, 1974). As a sim- 1 day's assays was freeze dried immediately before use. plification of the assay for choline acetyltransferase, the Incubation times were up to 30min (see text). scintillation mixture was used as the extraction solvent Tryptophan hydroxylase was assayed under similar conditions using 2 ~ M - ~ - M P H instead , of DMPH4 (TONG& (FONNUM, 1975). We describe here the application of procedures of this KAUFMAN,1975), 4-15 p~-~-[~H]tryptophan (1.17 x lo6 kind to the assay of tyrosine hydroxylase. The assays are d.p.m.) or 1 3 4 0 p ~ - ~ - [ ' ~ c ] t r y p t o p h a(9.2 n x lo4 d.p.m.) comparable in sensitivity with those described previously, and 4 pl-decarboxylase preparation. Incubations were for but they facilitate the management of large numbers of up to 60 min (see text). samples. Assays using Kalignost extractions are compared The decarboxylase was assayed under the conditions of for tyrosine hydroxylase, tryptophan hydroxylase, and aro- the tyrosine hydroxylase reactions using 1.0 to 800 p ~ matic L-amino acid decarboxylase. L-[~H]DOPA (1.7 x lo6 d.p.m.) and unlabelled tyrosine (final concentrations, G100 p ~ as , required). Alternatively, OXY (1.06 x lo6 5.M.O ~ M - D , L - ~ - ~ Y ~ T [3H]tryptophan MATERIALS AND METHODS d.p.m.) and 14.4 pM-unlabelled L-tryptophan were used for C3H]Water; ~-[3,5-~H]tyrosine; ~-[G-~H]tryptophan; assays under the conditions of the tryptophan hydroxylase ~-3,4-dihydroxy[ring-2,5,6-~H]phenylalaninereactions. Incubations were for 5 and 15 min, respectively. (C3H]D0PA); [3H]dopamine hydrochloride; ~ , ~ - 5 - h y d - All labelled substrates were freeze dried immediately before r~xy[G-~H]tryptophanand ~-[methylene-'~C]tryptophan use. The reactions were at 37°C in small glass tubes were obtained from the Radiochemical Centre, Amersham, Bucks. 6,7-Dimethyl-5,6,7,8-tetrahydropterinehydrochlor- (10 x 5 mm). To 10 pl samples of tissue homogenates were ide (DMPH,) and ~,~-6-methyl-5,6,7,8-tetrahydropterine added 10 p1 of the substrate mixture lacking only the hydroxylase. After incubation, the small tubes were placed (6-MPH4) were obtained from Calbiochem Ltd., and stored under nitrogen ( - 20°C). Activated alumina (acidic) in scintillation vials and the contents were washed out with 5ml of an ice cold solution of dopamine, or 5-HT from BDH Chemicals Ltd., was used without further treatment. All other reagents were of the highest purity avail- (0.25 mM), and ascorbic acid ( I mM) in 10 mM-sodium phosable. Solutions of DMPH4 or 6-MPH4, ferrous ammonium phate, pH 7.0. Acetonitrile (2 ml) containing 30 mg of sulphate, pargyline, dithiothreitol, dopamine, 5-HT and sodium tetraphenylboron was thoroughly mixed with the ascorbic acid were prepared immediately before use. resulting solution and 10 ml of scintillant (FONNUM, 1975) [3H]Tyrosine was purified before use by chroma- was added. After gentle shaking for 1 min, the vials were placed in a scintillation counter, allowed to stand for a tography on alumina and on Dowex and stored (-20°C) in absolute ethanol (COYLE,1972). minimum of 10min and counted (efficiencies: 3H, 24%; Aromatic L-amino acid decarboxylase was extracted I4C, 90%) within 12 h. This avoids quenching by coloured from pig kidneys, partially purified by ammonium sulphate oxidation products that appear after approx 24 h. 743
"
