Kinetic Properties Of Tyrosine Hydroxylase Research Articles

Inhibition of tyrosine hydroxylase by R and S enantiomers of salsolinol, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline.

Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.

Changes associated with early weaning in the activity of tyrosine hydroxylase in the caudate nucleus of the piglet

1. Early weaning in piglets is associated with changes in dopamine metabolism in the caudate nucleus. In particular, a decrease in the amount of the enzyme tyrosine hydroxylase was observed. 2. An analysis of the kinetic properties of tyrosine hydroxylase from the caudate nucleus of early-weaned piglets shows that there is a relative increase in the activity of the enzyme which may indicate a mechanism compensating for the decreased amount of enzyme. 3. The increase in activity is mediated through a decrease in the K m for tetrahydrobiopterin and is reversed, in vitro, when the tissue is homogenized at pH values greater than 8.0.

Monovalent cations and striatal tyrosine hydroxylase.

The kinetic properties of soluble tyrosine hydroxylase from rat striatum and the activation of the enzyme by the polyanion heparin were assessed as a function of the monovalent cations K+, Na+, tetramethylammonium (TMA+), and Tris. Substitution of K+ or Na+ for TMA+ or Tris can alter the kinetic properties of tyrosine hydroxylase in the absence of heparin the nature of the interaction of the enzyme with heparin and also the kinetic properties of the heparin-activated enzyme. The data suggest that monovalent cations can support unique conformational states of the enzyme.

Kinetic properties of tyrosine hydroxylase purified from bovine adrenal medulla and bovine caudate nucleus

Tyrosine hydroxylase ( l-tyrosine,tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) was highly purified from the soluble fraction of either bovine adrenal medulla or bovine caudate nucleus and the kinetic properties were compared using 6-methyltetrahydropterin or natural (6R)- l -erythro- tetrahydrobiopterin . Two enzyme fractions with different molecular weights from bovine adrenal medulla were separated on a DEAE-Sephacel column; the apparent molecular weights of the first and second enzyme fractions (Fraction I and Fraction II) were estimated to be 280 000 and 390 000, respectively, by Bio-Gel A-1.5 m chromatography. Fraction II was judged to be composed of several enzyme forms with different molecular weights. The molecular weight of the subunit of Fraction I was estimated to be 60 000 by SDS-polacrylamide slab gel electrophoresis. Therefore, the enzyme may be a tetramer. The kinetic properties of the low molecular weight form (Fraction I) and the high molecular weight form (Fraction II) were compared. When 6-methyltetradropterin was used as an artificial cofactor, Fraction II showed two distinct apparent K m values for the pterin cofactor, whereas Fraction I showed a single apparent K m value. Apparent K m values for tyrosine and oxygen were similar in the two enzyme preparations, and neither tyrosine nor oxygen was inhibitory. When (6R)- l -erythro- tetrahydrobiopterin was used as the natural cofactor, Fraction I showed two distinct apparent K m values for tetrahydrohiopterin in the presence of 217 μM oxygen and 50 μM tyrosine, but a single apparent K m value in the presence of 49 μM oxygen and 50 μM tyrosine. Tyrosine at 100 μM and oxygen at 94 μM were inhibitory on Fraction I with tetrahydrobiopterin as cofactor. These results show that two apparent K m values for natural (6R)- l -erythro- tetrahydrobiopterin cofactor can be observed only in air (217 μM oxygen), and that the different enzyme forms have different apparent K m values for the pterin cofactor. Tyrosine hydroxylase activity in bovine caudate nucleus was also separated into two fractions on a DEAE-Sepahcel column; the major peak was purified further and the apparent molecular weight was estimated to be 310 000 on a Bio-Gel A-1.5 m column. When natural (6R)- l-erythro- tetrahydrobiopterin was used as a cofactor, the enzyme showed a single apparent K m value for tetrahydropterin cofactor in the presence of 217 μM oxygen and 20 μM tyrosine. This result shows that tyrosine hydroxylase from caudate nucleus has a similar molecular weight but different kinetic properties compared with the enzyme from the adrenal medulla.

Kinetic properties of tyrosine hydroxylase with natural tetrahydrobiopterin as cofactor

A reproducible purification procedure of native tyrosine hydroxylase ( l-tyrosine, tetrahydropteridine : oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the soluble fraction of the bovine adrenal medulla has been established. This procedure accomplished a 90-fold purification with a recovery of 30% of the activity. This purified enzyme served for studying the kinetic properties of tyrosine hydroxylase using (6R)- l -erythro-1′,2′- dihydroxypropyltetrahydropterin [ (6R)- l -erythro- tetrahydrobiopterin ] as cofactor, which is supposed to be a natural cofactor. Two different K m values for tyrosine, oxygen and natural (6R)- l -erythro- tetrahydrobiopterin itself were obtained depending on the concentration of the tetrahydrobiopterin cofactor. In contrast, when unnatural (6S)- l -erythro- tetrahydrobiopterin was used as cofactor, a single K m value for each tyrosine, oxygen and the cofactor was obtained independent of the cofactor concentration. The lower K m value for (6R)- l -erythro- tetrahydrobiopterin was close to the tetrahydrobiopterin concentration in tissue, indicating a high affinity of the enzyme to the natural cofactor under the in vivo conditions. Tyrosine was inhibitory at 100 μM with (6R)- l -erythro- tetrahydrobiopterin as cofactor, and the inhibition by tyrosine was dependent on the concentrations of both pterin cofactor and oxygen. Oxygen at concentrations higher than 4.8% was also inhibitory with (6R)- l -erythro- tetrahydrobiopterin as cofactor.

A sensitive and inexpensive high-performance liquid chromatographic assay for tyrosine hydroxylase.

We describe a highly sensitive assay method for tyrosine hydroxylase (TH) using high-performance liquid chromatography with amperometric determination. This assay method could be applicable to any tissues with low enzyme activity, such as rat cerebellum. We also describe the kinetic properties of TH in rat cerebral cortex.

The activity of rat pineal and brain tyrosine hydroxylase during the daily cycle of light and darkness as determined by the modified 14CO2 assay method.

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a two-step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X-100 were as follows. The apparent Km values of the brain enzyme for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-L-erythro-biopterin (BPH4) as cofactor and for BPH4 with 62 microM-L-tyrosine as substrate were approximately 25 microM and 85 microM, respectively. The Km's for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-6-methylpterin (6MPH4) as cofactor and for 6MPH4 with 210 microM-L-tyrosine as substrate were 68 microM and 270 microM, respectively. The marked substrate inhibition by high concentrations of L-tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver-Burk plot of reciprocal velocity versus the reciprocal concentration of BPH4 with 62 microM-L-tyrosine as substrate deviated downward at a BPH4 concentration of about 100 microM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (6L- and 6D-isomers) of the tetrahydrobiopterin cofactor, with apparent Km for BPH4 of 23 microM and 1025 microM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.

Foot-shock stress accelerates non-striatal dopamine synthesis without activating tyrosine hydroxylase.

Electric foot-shock stress (20 min) increases DOPAC content in the frontal cortex (by about 80%) and in the nucleus accumbens (by 35%) but not in the striatum. However, foot shock stress failed to modify the kinetic properties of tyrosine hydroxylase (Vmax, Km for DMPH4 cofactor) in any of the above areas. Similar results were obtained in rats in which noradrenergic terminals in the n. accumbens and in the frontal cortex had been eliminated by injection of 6-OH-dopamine into the ascending dorsal noradrenergic bundle. The results support the hypothesis that limbic and cortical DA is involved in emotional states and indicate that DA synthesis may be regulated independently from changes in the kinetics properties of tyrosine hydroxylase.

Tyrosine hydroxylase regulation in rat striatal and olfactory tubercle slices—I: Activation induced by exposure to a calcium-free and high-magnesium medium

Slices from rat corpus striatum and olfactory tubercle were incubated for 15 min at 37° in Krebs-Ringer phosphate (KRP) medium or Ca 2+-free KRP medium both in the presence and absence of high Mg 2+ (12 mM). Tyrosine hydroxylase activity and kinetic parameters were determined in the 20,000 g supernatant fraction prepared from slices. The omission of Ca 2+ from the incubation medium resulted in a moderate increase in the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, and a significant increase in the activity of tyrosine hydroxylase isolated from olfactory tubercle slices. The presence of Mg 2+ (12 mM) in the Ca 2+-free KRP medium produced a further increase in the activity of the enzyme isolated both from striatal and olfactory tubercle slices. The per cent of stimulation of enzyme activity induced by incubating the slices in a Ca 2+-free, high-Mg 2+ KRP medium was maximal when assayed at pH 7.0. The Mg 2+-induced activation of tyrosine hydroxylase was antagonized by increasing the Ca 2+ concentration (3.75 to 15.0 mM) in the medium in which the slices were incubated. The direct addition of Mg 2+ (5–20 mM) to striatal and olfactory tubercle homogenates also resulted in an increase in the activity of tyrosine hydroxylase. The addition of high concentrations of Ca 2+ (10 mM) to the homogenates also resulted in an increase in enzyme activity but this effect was additive to that produced by Mg 2+ (10 mM). Incubation of striatal slices in a Ca 2+-free, high-Mg 2+ KRP medium produced changes in the kinetic properties of tyrosine hydroxylase. The apparent m of the enzyme for 6-methyl-5,6,7,8-tetrahydropterine HCl (6-MPH 4) was decreased significantly from 0.85 to 0.40 mM, with no significant change in the V max. However, the K i of the enzyme for dopamine (DA) was unchanged. Magnesium ions (12 mM) added to KRP-high K + (55 mM) medium blocked the activation of tyrosine hydroxylase induced by K +-depolarization of striatal slices. However, Mg 2+ (12 mM) addition to the incubation medium did not block but actually further increased the tyrosine hydroxylase activation that results after incubating striatal slices in a KRP medium enriched with dibutyryl cAMP (1 mM). Moreover, the stimulating effect on tyrosine hydroxylase observed when assays were conducted in the presence of Mg 2+, ATP and cAMP remained unchanged in homogenates prepared from slices previously incubated in a Ca 2+-free, high-Mg 2+ KRP medium. The results reported in this work clearly demonstrate that a kinetic activation of tyrosine hydroxylase in dopaminergic nerve terminals can occur under conditions of diminished extracellular Ca 2+ and blockade of transmitter release. Cyclic AMP does not seem to play a role in the tyrosine hydroxylase activation induced by incubation of slices in Ca 2+-free and high-Mg 2+ medium. The results support the hypothesis that the increase in DA biosynthesis observed in dopaminergic neurons after inhibition of impulse flow may result primarily as a consequence of a diminished entrance of Ca 2+ into the nerve terminal.

Effects of ovarian steroids on in vitro kinetic properties of tyrosine hydroxylase from rabbit oviducts.

The kinetic properties of tyrosine hydroxylase (TH) isolated from oviductal isthmuses of rabbits in different hormonal states were studied in order to determine whether the changes in norepinephrine (NE) metabolism in the oviduct observed previously (Kennedy and Marshall, 1977) were related to alterations in the activity of this rate limiting enzyme for NE synthesis. Ovariectomy reduced the reaction velocity (Vmax) of both TH and its pteridine cofactor, but did not change the affinity (Km) of the TH for either substrate or cofactor. Treatment of the castrates with estrogen or estrogen and progesterone returned Vmax to normal estrous levels. Therefore, the increase in NE content and turnover found previously in oviducts from castrates given ovarian steroids is related to an elevation of Vmax for both TH and cofactor and not to a change in Km. These actions of the ovarian steroids are reminiscent of those previously described for the effects of adrenocorticotropins on TH activity of the adrenal gland of hypophysectomized rats (Mueller et al., 1970) and represent another example of the influence of hormones on transmitter metabolism in the adrenergic nervous system.

The effect of castration, thyroidectomy and haloperidol upon the turnover rates of dopamine and norepinephrine and the kinetic properties of tyrosine hydroxylase in discrete hypothalamic nuclei of the male rat

Adult male rats were either castrated, thyroidectomized, or treated with haloperidol and the rates of turnover of dopamine (DA) and norepinephrine (NE) in the median eminence (ME), the arcuate and dorsomedial nuclei of the hypothalamus were estimated from the rate of decay of DA and NE concentrations as determined by radioenzymatic assay following blockade of catecholamine synthesis byalpha-methyl- p-tyrosine. The ME of animals similarly prepared was also examined for changes in the total activity and kinetic properties of tyrosine hydroxylase (TH). Four days following the administration of haloperidol (400 μg/kg) or 10 days after castration, there was a significant increase in the rate of turnover of DA but not NE in the ME accompanied by an increase in the V max but not K m for substrate or cofactor of TH. Furthermore, the administration of haloperidol to hypophysectomized rats also significantly increased the TH activity in the ME, indicating that such changes may occur independently of any changes in serum prolactin levels. Ten days after thyroidectomy, or three weeks after treatment with propylthiouracil, there was a significant increase in the turnover rate of DA in both the ME and dorsomedial nucleus but not in the arcuate nucleus. No changes in the turnover rates of NE in anu of the three areas were observed following thyroidectomy. In the ME, the increase in turnover of DA was accompanied by an increase in the total TH activity (V max) as well as a decrease in K m for tetrahydrobiopterin but not tyrosine. From these results 4 conclusions were drawn: (1) following haloperidol, castration, and thyroidectomy there are increases in the activity of dopaminergic terminals within the ME; (2) castration, haloperidol and thyroidectomy may influence the activity of dopaminergic terminals within the ME by different mechanisms; (3) changes in tyrosine hydroxylase and turnover of catecholamines within the ME may occur independently of changes in prolactin levels; and (4) local recurrent afferent circuits may exist in the arcuate nucleus region of the hypothalamus.

Activation of tyrosine hydroxylase in rat striatal slices by K+-depolarization— Effect of ethanol

Slices from rat corpus striatuum were incubated for 10 min at 37° in freshlyy oxygenated Krebs-Ringer phosphate (KRP) media or KRP-high K+ (55mM) media both in the presence and absence of ethanol (0.2 to 0.8%, w/v). Thereafter, the slices were homogenized and tyrosine hydroxylase activity and kinetic parameters were determined in the 105,000 g supernatant fraction. The presence of K+ (55 mM) in the incubation media increased about 3-fold the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, when compared to that found in striatal slices incubated in normal KRP media. Incubation of striatal slices in a KRP-high K+ media also produced changes in the kinetic properties of tyrosine hydroxylase. The Km of the enzyme for 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine HCl (DMPH4) was decreased from 0.82 to 0.09 mM and the Ki of the enzyme for dopamine (DA) was increased from 0.13 to 3.52 mM. Ethanol (0.2 to 0.8%, w/v) added directly to the KRP-high K+ media markedly blocked the K+-induced activation of tyrosine hydroxylase as well as the kinetic alterations in the enzyme observed after K+-depolarization of the striatal slices. In contrast, the presence of ethanol did not modify the activity and kinetic characteristics of tyrosine hydroxylase isolated from slices incubated in normal KRP media. The results reported in this work suggest that the increase in DA synthesis observed in striatal slices after K+-depolarization might be mediated in part via an allosteric activation of tyrosine hydroxylase. This activation appears to be mediated by an increase in the affinity of the enzyme for the pterin cofactor and a decreased affinity for the end-product inhibitor DA. Also, the blocking effect of ethanol upon the kinetic activation of tyrosine hydroxylase after K+-depolarization seems to offer a likely explanation for the inhibitory effect of ethanol on K+-induced increase in DA synthesis reported recently by Gysling et al. (Biochem. Pharmac.25, 157 (1976)).

Dopaminergic neurons—Alteration in the kinetic properties of tyrosine hydroxylase after cessation of impulse flow

Studies in vitro conducted on striatal tyrosine hydroxylase have demonstrated that addition of calcium chelators, such as EGTA, results in an alteration in the kinetic properties of tyrosine hydroxylase which can be completely reversed by addition of calcium to the incubation medium. Inhibition of impulse flow in the nigro-neostriatal pathway by either pharmacological or mechanical techniques causes the isolated striatal tyrosine hydroxylase to behave kinetically like the enzyme treated in vitro with EGTA. Tyrosine hydroxylase, isolated from the striatum of rats in which impulse flow has been interrupted in the dopamine pathways, has an increased affinity for both substrate and pterin cofactor and a reduced affinity for the end-product inhibitor, dopamine. The latter change is most dramatic; the K i of the enzyme for dopamine. increases more than 700-fold. These alterations in the kinetic properties of tyrosine hydroxylase can also be completely reversed by addition of calcium to the incubation medium. In rats with a pharmacological blockade of impulse flow induced by administration of γ-hydroxybutyrate, subsequent administration of dopamine receptor stimulants causes the isolated tyrosine hydroxylase to have properties similar to those observed for the enzyme prepared from untreated rats. Administration of dopamine receptor blockers prevents the dopamine receptor stimulants from altering the kinetic changes in tyrosine hydroxylase induced by a cessation of impulse flow. These data are discussed in terms of the possible role presynaptic receptors and calcium fluxes may play in the short-term regulation of tyrosine hydroxylase.

Tyrosine hydroxylase: activation by nerve stimulation.

The synthesis of the sympathetic neurotransmitter, norepinephrine, is accelerated by electrical stimulation of the guinea pig vas deferens. The molecular mechanism responsible for this enhanced formation of transmitter is unknown but has been attributed to an increase in the activity of tyrosine hydroxylase (EC 1.14.16.2; tyrosine 3-monooxygenase) during nerve stimulation. In the present experiments, we found that crude preparations of tyrosine hydroxylase isolated from guinea pig vasa deferentia that were electrically stimulated or depolarized by potassium show an increase in activity compared with enzyme obtained from untreated paired control tissues. This increase in activity is partially antagonized by addition of the Ca(++) chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), to the assay medium, and can be completely blocked if Ca(++) is removed from the potassium-rich medium used to depolarize the intact tissue, before preparation of the enzyme. A similar increase in enzyme activity occurs when Ca(++) ions are added directly to enzyme prepared from untreated vasa deferentia. In this instance, the activation is completely reversed by EGTA. The increase in activity produced by addition of Ca(++) to the isolated enzyme or by electrical stimulation or potassium depolarization of the tissue before isolation of the enzyme appears to be mediated by changes in the kinetic properties of tyrosine hydroxylase. All treatments appear to activate tyrosine hydroxylase by causing an increase in its affinity for substrate and pteridine cofactor and by decreasing its affinity for the end-product inhibitor, norepinephrine. These results provide direct evidence that the enhanced formation of norepinephrine seen during stimulation of sympathetically innervated tissues arises from an activation of tyrosine hydroxylase. The fact that the activation produced by nerve stimulation is mimicked by Ca(++) ions raises the intriguing possibility that the influx or mobilization of Ca(++) that accompanies nerve stimulation and that is intimately involved in release of transmitter may also participate in the activation of tyrosine hydroxylase.