Km Of Tyrosine Hydroxylase Research Articles

Abnormal norepinephrine metabolism in rat brain synaptosomes in phosphate depletion.

Abnormalities in the function of the central nervous system exist in phosphate depletion (PD). It is possible that this is due to an adverse effect of PD on the metabolism of neurotransmitters, such as norepinephrine (NE), in brain synaptosomes. We examined the effects of PD, produced by restriction of dietary phosphate intake on NE metabolism of brain synaptosomes. Synaptosomes from PD rats had significantly reduced NE content, uptake and release, elevated Km, but normal Vmax of tyrosine hydroxylase, normal Km and Vmax of monoamine oxidase, elevated resting levels of cytosolic calcium ([Ca2+]i), higher delta [Ca2+]i in response to KCl, higher delta [Ca2+]i/basal [Ca2+]i ratio, lower ATP content and reduced activity of Na(+)-K(+)-ATPase as compared to synaptosomes from pair-weighed rats. Treatment of PD rats with verapamil corrected all the synaptosomal derangements except for the elevated Km of tyrosine hydroxylase and NE content. Verapamil did not affect the metabolism of PW rats. The data demonstrate that PD causes significant derangements in NE metabolism of brain synaptosomes. Observations in the present study and in others indicate that these derangements in NE metabolism are due to the PD-induced abnormalities in the homeostasis of synaptosomal [Ca2+]i, ATP and phospholipids and in the activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase.

Phosphorylation of Rat Tyrosine Hydroxylase and Its Model Peptides In Vitro by Cyclic AMP‐Dependent Protein Kinase

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.

Short and long term regulation of catecholamine biosynthetic enzymes by angiotensin in cultured adrenal medullary cells. Molecular mechanisms and nature of second messenger systems.

The purpose of this study was to examine the effects of angiotensin on the enzyme activities and gene expression of two catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT), in bovine adrenal medullary (AM) cells. Short term (15 min) incubation of cultured AM cells with 2 nM [Sar1]angiotensin II (s1-AII) did not increase basal secretion of catecholamines; however, longer incubations (3, 24, or 72 h) produced 4-10-fold increases. To determine whether angiotensin affects synthesis of catecholamines, the activities of TH and PNMT were examined. Incubation with s1-AII (15-30 min) decreased the Km of TH for its biopterine cofactor [6R)-5,6,7,8-tetrahydro-1-biopterin dihydrochloride (BH4] without affecting the Vmax, suggesting activation of TH. After long term incubation (72 h) the Km value was identical to that of control, while increases in the apparent Vmax were observed. PNMT activity was unaffected during a 30-min treatment with s1-AII; however, 2-fold increases occurred after a 48-72-h incubation. s1-AII (24 h) increased the relative abundance of TH and PNMT mRNAs, suggesting that the long term increase in enzyme activities reflected increased expression of TH and PNMT genes. Maximal increases were observed at 2 nM s1-AII and the changes were antagonized by saralasin. Induction of TH mRNA by s1-AII was additive to the effects of veratridine or forskolin indicating that effects of angiotensin were not due to membrane depolarization or increased cyclic AMP levels. Incubation with Ca2+ ionophore A23187 increased TH and PNMT mRNA levels in AM cells raising the possibility that the increase in cellular [Ca2+] could mediate effects of angiotensin. Angiotensin-induced increases in TH and PNMT mRNA were inhibited by nifedipine indicating involvement of voltage-dependent Ca2+ channels. In addition, the increases in TH, but not PNMT mRNA, were antagonized by dantrolene, which inhibits mobilization of Ca2+ from intracellular stores. Calmodulin involvement was suggested by the inhibition of s1-AII induced changes in mRNA with 1 microM calmidazolium.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of TRH analog (DN-1417) on tyrosine hydroxylase activity in mesocortical, mesolimbic and nigrostriatal dopaminergic neurons of rat brain.

Tyrosine hydroxylase (TH) was assayed in eight regions of rat brain following repeated treatment with a TRH analog, DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide). Repeated DN-1417 treatment (20 mg/kg/day, IP) for 7 days increased TH activity in the ventral tegmental area and decreased in the prefrontal cortex polar, medial and lateral fields and olfactory tubercles. No significant change in TH activity was found in the nucleus accumbens, striatum and substantia nigra. Kinetic analysis showed that the increased TH activity in the ventral tegmental area was due to an increase in Vmax, but not a change in the apparent Km of TH for a cofactor, 6-methyl-tetrahydropteridine. When TH was assayed at a suboptimal pH and in the presence of a subsaturating cofactor, the striatal TH was activated significantly after DN-1417. In the prefrontal cortex medial field, nucleus accumbens and olfactory tubercles, TH activity assayed under the suboptimal condition was not modified by DN-1417 treatment. These results suggest an intimate involvement of central dopaminergic systems in the actions of DN-1417.

Stimulation of retinal dopamine biosynthesis in vivo by exogenous tetrahydrobiopterin: relationship to tyrosine hydroxylase activation

Intravitreal injection of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), increases 3,4-dihydroxyphenylalanine (DOPA) accumulation in retinas of dark-adapted rats, as does exposure to light. In contrast, BH4 had no significant effect on DOPA accumulation in retinas of light-exposed rats. The levels of endogenous retinal BH4 and the uptake of injected BH4 into the retinal tissue were not affected by light exposure. These data indicate that TH is not saturated with endogenous BH4 in the retinas of dark-adapted rats. In addition, the observations support the interpretation that the decrease in apparent Km of TH for the cofactor in response to light exposure is of sufficient magnitude to allow near saturation of TH by endogenous BH4 and, thus, is causally related to the increase of dopamine biosynthesis in response to short-term photic stimulation.

Effects of db cAMP on tyrosine hydroxylase activity of ganglia and nerve endings.

Preincubation of intact superior cervical ganglia or nictitating membrane for 2 h with dibutyryl cyclic AMP (db cAMP) increased the hydroxylation of tyrosine. This effect was not blocked by the protein synthesis inhibitor, cycloheximide. The Km of tyrosine hydroxylase for the substrate, tyrosine, and for the cofactor, reduced pteridine, were decreased by db cAMP. There were no changes in the Vmax of the enzyme. The inhibitory potency of noradrenaline on the hydroxylation of tyrosine was also decreased. Thus an inductive effect may be ruled out. The activation of the enzyme was only observed when the tissues were preincubated with the db cAMP and not when the cyclic nucleotide was added to the isolated enzyme. Preincubation of cervical ganglia for 4 h with db cAMP increased activity of decarboxylase and monoamine oxidase in tissue homogenates without changing the tyrosine hydroxylase activity.

Thyroid cold acclimation influences on norepinephrine metabolism in brown fat.

Norepinephrine turnover rates and tyrosine hydroxylase activities were determined in the interscapular brown fat pad of the rat during cold acclimation, hyperthyroxinism, and after thyroidectomy. Rats were cold acclimated by placement in a cold room, one rat to a cage, for a period of 6 wk. Hyperthyroxinism was induced by daily subcutaneous injections of L-thyroxine (1 mg/kg) for 6 days. Norepinephrine turnover rate and enzyme activity were determined at the end of each experimental period and at 8 wk after thyroidectomy. The rate of norepinephrine turnover increased during cold acclimation and hyperthyroxinism and decreased after thyroidectomy. Cold acclimation resulted in a significant increase in tyrosine hydroxylase activity, whereas no significant effect on enzyme activity was observed in hyperthyroxinism or after thyroidectomy. None of the conditions produced a change compared to controls in the apparent Km of tyrosine hydroxylase for L-tyrosine. Cold acclimation resulted in a significant decrease in the apparent Km of tyrosine hydroxylase for pterin cofactor, whereas thyroxine treatment and thyroidectomy had no effect.

Regulation of catecholamine synthesis in rat brain synaptosomes.

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent Km of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10−6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10−7m; noradrenaline, 25% inhibition at 5 ± 10−6m;and serotonin, 30% inhibition at 10−5m. The catecholamine‐induced inhibition of synthesis was antagonized by pre‐incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra‐synaptosomal amine levels, and that short‐term exposure to depolarizing concentrations of potassium can increase synthesis.