Tyrosine Hydroxylase Activation and Inactivation by Protein Phosphorylation Conditions

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, catalyzes the conversion of tyrosine to DOPA, Cyclic AMP-dependent protein phosphorylation conditions alter tyrosine hydroxylase activity in rat striatal homogenates. In agreement with other laboratories, we find that short-term pre-incubation (3 min) of extracts under phosphorylating conditions (Mg . ATP, cAMP) increases enzyme activity two- to tenfold over control as measured during a subsequent 15-min assay. We now report that preincubation under phosphorylating conditions for longer periods (30 min) results in a loss of activity to levels equal to or below that of the control enzyme. Addition of purified bovine brain protein kinase catalytic subunit and Mg . ATP enhances activation and increases the rate of inactivation. To demonstrate that inactivation is not associated with proteolytic degradation or irreversible denaturation, the inactivated form of the enzyme can be reactivated. The protein kinase inhibitor protein decreases the activation process and prevents inactivation of the enzyme to below control values. The sedimentation coefficient is not changed by phosphorylation conditions (S = 8.8 +/- 0.1). Although the apparent Km of the enzyme for the 6-methyltetrahydropterine (6-MPH4) cofactor is reduced (0.86 mM, control; 0.32 mM, activated), it is also reduced in the inactivated form (0.38 mM). The Ki for dopamine is increased from 4.5 microM for the control to 28 microM for the activated enzyme, whereas the inactivated form of the enzyme exhibits a Ki of 10 microM. Removal of catecholamines by gel filtration fails to alter activity and the apparent cofactor Km. Moreover, both the activated and the inactivated states persist following gel filtration. It therefore appears that the activation-inactivation process is not mediated solely by the modulation of enzyme feedback inhibition or changes in the Km for 6-MPH4. We also describe a coupled decarboxylase assay in which labeled dopamine is resolved from the precursors tyrosine and DOPA by low-voltage paper electrophoresis.     

Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α -methyl- p -tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.     

Synthesis of l-3,4-Bihydroxyphenylalanine by Tyrosine Hydroxylase cDNA-Transfected C6 Cells: Application for Intracerebral Grafting

In the present study, we obtained genetically manipulated nonneuronal cells which synthesize a catecholamine precursor for future use in intracerebral grafting. Human type 1 tyrosine hydroxylase (TH; EC 1.14.16.2) cDNA was inserted into eukaryotic expression vector pKCRH2 and was co-transfected into C6 cells with plasmid pSV2neo. Expression of the TH minigene was screened by immuno-histochemical staining with TH antibody and immunoblot-ting analysis. Several clones of the C6 transfectahts that produce TH molecules were obtained. These cells showed TH activity, and the product, L-3,4-dihydroxyphenylalanine (L-DOPA), was detected intracellulary due to the ajbsence of L-amino acid decarboxylase (EC 4.1.1.28) activity. It was found that a large amount of L-DOPA was released from the cells into the culture medium. These transfectants were transplanted into rat brain, and the expression of TH was examined immunohistochemically. On the 10th day following transplantation, a mass of C6 cells which was heavily stained with TH antibody was observed in the brain. These findings may provide us with an opportunity to investigate the effects of intracerebral transplantation of nonneuronal cells that produce catecholamine or its precursor.     

大鼠酪氨酸羟化酶基因在肌母细胞中稳定表达

用电穿孔法将大鼠酪氨酸羟化酶（Ｔｙｒｏｓｉｎｅｈｙｄｒｏｘｙｌａｓｅ,ＴＨ）基因转染大鼠Ｌ－６ＴＧ成肌细胞株,经ＰＣＲ检测、免疫组织化学和荧光组织化学检测证明,ＴＨ基因能在细胞内稳定整合和表达,并在辅因子存在时将酪氨酸转化为多巴．移植于大鼠纹状体后可成活并表达ＴＨ。     

Thermal Denaturation of Native Striatal Tyrosine Hydroxylase: Increased Thermolability of the Phosphorylated Form of the Enzyme

Tyrosine hydroxylase was purified from bovine corpus striatum. The native enzyme had a half-life of 15 +/- 3 min at 50 degrees C. Phosphorylation of tyrosine hydroxylase with protein kinase purified from both corpus striatum and heart activated the enzyme, but activity was rapidly lost with additional preincubation of the enzyme at 30 degrees C. Thermal denaturation studies indicated that phosphorylated tyrosine hydroxylase had a half-life of 5 +/- 2 min at 50 degrees C     

Monovalent Cations and Striatal Tyrosine Hydroxylase

Abstract: 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.     

Tyrosine Hydroxylase Is Inactivated by Catechol-Quinones and Converted to a Redox-Cycling Quinoprotein

Quinone derivatives of DOPA, dopamine, and N-acetyldopamine inactivate tyrosine hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. The parent catechols are inert in this capacity. The effects of the catecholquinones on tyrosine hydroxylase are prevented by antioxidants and reducing reagents but not by scavengers of hydrogen peroxide, hydroxyl radicals, or superoxide radicals. Quinone modification of tyrosine hydroxylase modifies enzyme sulfhydryl groups and results in the formation of cysteinyl-catechols within the enzyme. Catecholquinones convert tyrosine hydroxylase to a redox-cycling quinoprotein. Quinotyrosine hydroxylase causes the reduction of the transition metals iron and copper and may therefore contribute to Fenton-like reactions and oxidative stress in neurons. The discovery that a phenotypic marker for catecholamine neurons can be converted into a redox-active species is highly relevant for neurodegenerative conditions such as Parkinson's disease.     

Antibodies to a Synthetic Peptide Corresponding to a Ser-40-Containing Segment of Tyrosine Hydroxylase: Activation and Immunohistochemical Localization of Tyrosine Hydroxylase

A peptide corresponding to position 32-47 in tyrosine hydroxylase was synthesized (TH-16) and polyclonal antibodies against this peptide were raised in rabbits (anti-TH-16). The effects of anti-TH-16 on modulation of tyrosine hydroxylase activity were investigated. Anti-TH-16 enhanced the enzymatic activity in a concentration-dependent manner, and the antigen TH-16 inhibited the stimulatory activity of the antiserum in a concentration-dependent manner. The activated enzyme had a lower Km app for the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and a higher Vmax app than the nonactivated enzyme. Anti-TH-16 was characterized further by its ability to immunoprecipitate the enzyme activity by labeling tyrosine hydroxylase after Western blotting and by immunohistochemical labeling of catecholaminergic neurons. Anti-TH-16 did not block activation of tyrosine hydroxylase by phosphorylation catalyzed by cyclic AMP-dependent protein kinase. Exposure of the enzyme to anti-TH-16 and subsequent phosphorylation of the enzyme resulted in a greater activation of the enzyme than the sum of activation produced by these two treatments separately. However, the activation was less than additive when the enzyme was first phosphorylated and subsequently exposed to anti-TH-16. The present study demonstrates the utility of anti-TH-16 in investigating the molecular aspects of the enzyme activation.     

Mutational Analysis of Substrate Inhibition in Tyrosine Hydroxylase

Abstract: Substrate inhibition in tyrosine hydroxylase (TH) was analyzed by deletion mutagenesis. The deletion mutant TH 156/456 was the smallest section of TH to retain substrate inhibition. The TH 156/456 was monomeric, and so multimer formation does not play a role in substrate inhibition in TH. Further deletion at the N terminus to residue 169 produced a TH molecule with no substrate inhibition but high activity. A mutagenic scan of this region showed that mutations at Trp¹⁶⁶ were responsible for this phenotype. A screen of a library of TH molecules containing random mutations identified three other mutants that had lost substrate inhibition but retained high activity. The results in this report are consistent with a model in which substrate inhibition acts through an allosteric mechanism.     

Dopamine Autoreceptors Modulate the Phosphorylation of Tyrosine Hydroxylase in Rat Striatal Slices

The hypothesis that dopamine (DA) autoreceptors modulate the phosphorylation of tyrosine hydroxylase (TH; EC 1.14.16.2) was investigated in rat striatal slices. Tissue was prelabeled with 32P inorganic phosphate, and TH recovered by immunoprecipitation with anti-TH rabbit serum. The TH monomer was resolved on sodium dodecyl sulfate polyacrylamide gels, and the extent of phosphorylation was determined by scanning densitometry of autoradiographs. Depolarization of striatal slices with 55 mM K+ markedly increased the incorporation of 32P into several proteins, including the TH monomer (Mr = 60,000). A similar increase in TH phosphorylation occurred in response to the adenylate cyclase activator forskolin and the cyclic AMP analog dibutyryl cyclic AMP. An increase in TH phosphorylation was not observed in response to the D1-selective agonist SKF 38393. The D2-selective DA autoreceptor agonist pergolide decreased the phosphorylation of TH below basal levels and blocked the increase in phosphorylation elicited by 55 mM K+. The inhibitory effect of pergolide was antagonized by the D2-selective antagonist eticlopride. Changes observed in the phosphorylation of TH were mirrored by changes in tyrosine hydroxylation in situ. These observations support the hypothesis that a reduction in TH phosphorylation is the mechanism by which DA autoreceptors modulate tyrosine hydroxylation in nigrostriatal nerve terminals.     

Central Regulation of Adrenal Tyrosine Hydroxylase: Effect of Induction on Catecholamine Levels in the Adrenal Medulla and Plasma

The effect of induction of adrenal tyrosine hydroxylase (TH) by various centrally acting drugs on catecholamine levels in adrenal and plasma was investigated in rats. All the drugs tested, namely oxotremorine, Piribedil, B-HT 920, and HA-966, produced significant increases in adrenal dopamine content and plasma epinephrine level. Denervation of the adrenal abolished the increase in adrenal dopamine as it did the induction of tyrosine hydroxylase. The results suggest that the induced increase of adrenal TH activity, as mediated by certain drugs, results in an elevation of the plasma epinephrine level and that the adrenal dopamine content is a better indicator of the catecholamine-synthesizing capacity of the adrenal medulla than are the other catecholamines.     

Isolation and Characterization of Ovine Tyrosine Hydroxylase mRNA

Abstract: Tyrosine hydroxylase (TH) cDNA has been characterized in rodents and primates, but only a few studies have been developed in ungulates, except in cows. Because sheep is a species used for many physiological studies, it was of interest to clone TH cDNA in this species. Ovine TH cDNA was purified from a library of sheep adrenal glands. The entire cDNA was 1,721 bp long. It presented a higher percentage of similarity with bovine TH cDNA (93%) than with rodent cDNAs (75%). The deduced amino acid sequence was 490 amino acids long and had 96% similarity with the bovine amino acid sequence. The entire cDNA and different fragments obtained with endonuclease restriction enzymes were cloned in plasmid pUC 18 and were labeled with ³⁵S-dATP to detect TH mRNA by in situ hybridization. Strong labelings were observed on adrenal medulla and on noradrenergic and dopaminergic neurons in the sheep but also in the cow and pig. This labeling matched completely TH immunohistochemical staining obtained on the same sections with anti-TH antibodies. Ovine TH cDNA is a useful tool to study the variations of TH mRNA levels in sheep catecholaminergic neurons.     

小鼠酪氨酸羟化酶启动子序列分析

目的对小鼠酪氨酸羟化酶（tyrosine hydroxylase,TH）启动子进行序列分析,研究启动子不同区域对下游基因表达调控能力。方法高保真PCR分别扩增出450、1500、2000、2400、3400bp TH启动子片段置换pcDNA3中CMV启动子,并在多克隆位点插入EGFP报告基因,重组后质粒分别瞬时转染MN-9D细胞（TH^＋）和ECV细胞（TH^-）,流式细胞仪观察EGFP基因在细胞内表达情况。结果转染后MN-9D细胞中EGFP阳性率分别为8.01%、7.97%、7.85%、7.72%、7.74%,差异无显著性。转染ECV细胞（TH^-）中,EGFP阳性率分别为6.51%、6.35%、6.71%、0.89%、1.02%。3400bp、2400bp启动子片段在TH^-细胞中调控能力受到明显抑制。结论上述启动子片段均有调控基因表达能力,初步证明TH启动子中特异性调控区域在2400-2000bp范围内。     

Antibodies to a Segment of Tyrosine Hydroxylase Phosphorylated at Serine 40

Abstract: A synthetic peptide corresponding to residues 32–47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser⁴⁰ and used to generate antibodies in rabbits. Reactivity of the anti-pTH_32–47 antibodies with phospho- and dephospho-Ser⁴⁰ forms of TH protein and peptide TH_32–47 was compared with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH_32–47 was more reactive with the phospho-TH than with the dephospho-TH forms. Conversely, antibodies against the nonphosphorylated peptide reacted preferentially with the dephospho-TH forms. In western blots, labeling of the ∼60-kDa TH band by anti-pTH_32–47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10–100 ng/lane. In blots of supernatants prepared from striatal synaptosomes, addition of a phosphatase inhibitor was necessary to discern labeling of the TH band with anti-pTH_32–47. Similarly, anti-pTH_32–47 failed to immunoprecipitate TH activity from supernatants prepared from untreated tissues, whereas prior treatment with either 8-bromoadenosine 3',5'-cyclic monophosphate or forskolin enabled removal of TH activity by anti-pTH_32–47. Lastly, in immunohistochemical studies, anti-pTH_32–47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat brain.