A New Enzyme Species of p-Hydroxybenzoate Hydroxylase during Oxygenating Process Observed by the Stopped-flow Method

Sulfite ion (HSO₃ ^-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO₃ ^- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO₃ ^-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO₃ ^- on cytochrome c oxidase activity were studied with the plasma membranes of HSO₃ ^--resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO₃ ^-. To investigate the inhibition mechanism of HSO₃ ^- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO₃ ^-. In contrast, the same concentration of HSO₃ ^- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO₃ ^- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.     

Enzyme-Substrate Complex of p-Hydroxybenzoate Hydroxylase; One of the Activated Intermediates of the Overall Reaction

The transglucosidation reaction of brewer’s yeast α-glucosidase was examined under the co-existence of l-sorbose and phenyl-α-glucoside. As the transglucosidation products, three kinds of new disaccharide were chromatographically isolated. It was presumed that these disaccharides consisting of d-glucose and l-sorbose were 1-O-α-d-glucopyranosyl-l-sorbose ([α]_D+89.0), 3-O-α-d-glucopyranosyl-l-sorbose ([α]_D+69.1) and 4-O-α-d-glucopyranosyl-l-sorbose ([α]_D+81.0). The principal product formed in the enzyme reaction was 1-O-α-d-glucopyranosyl-l-sorbose.     

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.     

Hydroxylase activity of immobilized haemoglobin

Until now, hydroxylation of substrates for practical applications has been mostly carried out by chemical and microbial processes. The hydroxylase activity of haemoglobin could be of great help for this purpose. Hydroxylation of aniline by haemoglobin immobilized as cross-linked soluble polymers and insoluble particles was studied. Activity yields after immobilization as well as kinetic constants were estimated. Hydroxylase activities similar to those of liver microsomal cytochrome P-450 activities were obtained.     

Physiological Characterization of Pseudomonas putida DOT-T1E Tolerance to p-Hydroxybenzoate

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.     

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.     

Structural Effects on Arthrobacter Methylene Hydroxylase Activity

Arthrobacter 4-44-2 (ATCC 25581), capable of subterminal oxidation of n-hexadecane to 2-, 3-, and 4-alcoholic and ketonic products, was examined for the ability of this methylene hydroxylase capability to be induced and repressed and for structural relationships influencing methylene function oxidation. Induction was best carried out by use of n-alkanes from 10 to 16 carbons in length and was especially strong with methylcyclohexane among cyclic compounds tested. Induction was not observed with several related alcohols, 1-unsaturated compounds, or methoxy and ethoxy compounds tested. After induction, n-alkanes 14 and 16 carbons in length were transformed to the corresponding internal oxidation products; however, no activity was observed with even-carbon alkanes of shorter chain length. Hexadecene-1 and all alcohols tested, including cyclododecanol, were transformed to corresponding ketonic or aldehydic products. Cyclic compounds tested, including cyclododecane, were not oxidized by induced cells, suggesting that a methyl group plays a role in orientation of the substrate for the methylene hydroxylation but that the methyl function was not as critical after completion of the hydroxylation step regardless of structural configuration. Acetate strongly repressed induction of n-hexadecane methylene hydroxylase activity. Inducibility of methylene hydroxylase activity was confirmed by use of cell-free systems with methylcyclohexane as an inducer. A stimulation of methylene hydroxylase activity by addition of reduced pyridine nucleotides and ferrous ion was indicated.     

间羟苯甲酸4－羟化酶纯化及部分性质研究

经超声破碎、硫酸铵分级沉淀、凝胶过滤、磷酸钙胶层析和离子交换层析等步骤, 从Comamonas testosteroni菌株中获得了SDS-PAGE单一条带, 相对分子质量为62×10³的间羟苯甲酸4-羟化酶比活提高21倍, 产率为30%.此酶为FAD加单氧酶, 催化间羟苯甲酸转变为3,4二羟苯甲酸.     

Chronic Cocaine Administration Increases CNS Tyrosine Hydroxylase Enzyme Activity and mRNA Levels and Tryptophan Hydroxylase Enzyme Activity Levels

Cocaine is an inhibitor of dopamine and serotonin reuptake by synaptic terminals and has potent reinforcing effects that lead to its abuse. Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting steps in dopamine and serotonin biosynthesis, respectively, and are the subject of dynamic regulatory mechanisms that could be sensitive to the actions of cocaine. This study assessed the effects of chronic cocaine on brain TH and TPH activities. Cocaine was administered (0.33 mg/infusion, i.v.) to rats for 7 days every 8 min for 6 h per day. This administration schedule is similar to patterns of self-administration by rats when given ad libitum access to this dose. This chronic, response-independent administration increased TH enzyme activity in the substantia nigra (30%) and ventral tegmental area (43%). Moreover, TH mRNA levels were also increased (45 and 50%, respectively). In contrast to the enzymatic and molecular biological changes in the cell bodies, TH activity was unchanged in the terminal fields (corpus striaturn and nucleus accumbens). Similarly, TPH activity was increased by 50% in the raphe nucleus (serotonergic cell bodies). In summary, the chronic response-independent administration of cocaine produces increases in the expression of TH mRNA and activity in both the cell bodies of motor (nigrostriatal) and reinforcement (mesolimbic) dopamine pathways. These increases are not manifested in the terminal fields of these pathways.     

l-DOPA Is a Substrate for Tyrosine Hydroxylase

Abstract: In the presence of thiols, tyrosine hydroxylase (TH) oxidizes l -dihydroxyphenylalanine ( l -DOPA) with a specific activity of up to 140 nmol min⁻¹ mg⁻¹ at 37°C and pH 7.0, which is ∼12–50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2-mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of l -DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.     

Tyrosine Hydroxylase Inactivation Following cAMP-Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho-diesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co-substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos-phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.     

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.     

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.     

Presence of Kynurenine Hydroxylase in Developing Rat Brain

Abstract: Kynurenine-3-hydroxylase, an enzyme that is part of the degradative pathway for tryptophan, was present in the cerebral cortex of neonatal rats and exhibited a K_m , for L-kynurenine close to that of the liver enzyme. This enzyme was enriched in mitochondrial fractions isolated from cerebral cortices of neonatal rats by Ficoll-sucrose gradient centrifugation, with some activity also present in synaptosomal fractions probably due to the mitochondrial content of synaptosomes since cytochrome c oxidase, another mitochondrial enzyme, had a similar distribution in the gradient. Kynurenine hydroxylase as well as monoamine oxidase, another mitochondrial enzyme, had increased specific activities in synaptosomal fractions isolated from 14-day-old rats compared to fractions from 8-day-old rats. Hypothyroidism, induced on the day of birth, resulted in increased activities of kynurenine hydroxylase and monoamine oxidase in synaptosomal fractions isolated from 14-day-old rats.     

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。