Mechanism of action of salsolinol on tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in dopamine synthesis. Dopamine regulates TH as an end-product inhibitor through its binding to a high and low affinity site, the former being abolished by Ser40 phosphorylation only, and the latter able to bind and dissociate according to intracellular dopamine levels. Here, we have investigated TH inhibition by a dopamine metabolite found in dopaminergic brain regions, salsolinol (SAL). SAL is known to decrease dopamine in the nigrostriatal pathway and mediobasal hypothalamus, and to also decrease plasma catecholamines in rat stress models, however a target and mechanism for the effects of SAL have not been found. We found that SAL inhibits TH activity in the nanomolar range in vitro, by binding to both the high and low affinity dopamine binding sites. SAL produces the same level of inhibition as dopamine when TH is non-phosphorylated. However, it produces 3.7-fold greater inhibition of Ser40-phosphorylated TH compared to dopamine by competing more strongly with tetrahydrobiopterin, the cofactor of this enzymatic reaction. SAL’s potent inhibition of phosphorylated TH would prevent TH from being fully activated to synthesise dopamine.     

Tyrosine Hydroxylase from Bovine Striatum: Catalytic Properties of the Phosphorylated and Nonphosphorylated Forms of the Purified Enzyme

Abstract: The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH₄) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH₄), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in V _max. At pH 7.0, phosphorylation caused increased activation of TH by increasing V _max as well as reducing the K _m for cofactor. The K _m for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in K _m for tyrosine at any pH or with any cofactor studied, although the K _m for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH₄ than with BH₄ as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

THE INHIBITION OF PHENYLALANINE AND TYROSINE HYDROXYLASES BY HIGH OXYGEN LEVELS

Abstract— The K _m for oxygen for rat liver phenylalanine hydroxylase depended on the structure of the reduced pterin cofactor. When the synthetic cofactor, 6,7-dimethyltetrahydropterin, was employed, the apparent K _m for oxygen was 20%. When the natural cofactor, tetrahydrobiopterin, was used, the apparent K _m for oxygen was 0.35 %. Substrate inhibition (40 per cent inhibition at 43% oxygen) was observed with the natural cofactor but not with the synthetic cofactor. Oxygen also caused substrate inhibition with bovine adrenal medulla and brain tyrosine hydroxylases. The inhibition was more dramatic in the presence of the natural cofactor than with the synthetic cofactor. Substrate inhibition by oxygen of brain tyrosine hydroxylase may explain the lowered brain levels of norepinephrine and dopamine observed after treatment of animals with hyperbaric oxygen.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Effects of temperature, body size and feeding on rates of metabolism in young‐of‐the‐year haddock

The mean rate of oxygen consumption (routine respiration rate, R _R, mg O₂ fish⁻¹ h⁻¹), measured for individual or small groups of haddock Melanogrammus aeglefinus (3–12 cm standard length, L _S) maintained for 5 days within flow‐through respiratory chambers at four different temperatures, increased with increasing dry mass ( M _D). The relationship between R _R and M _D was allometric ( R _R = α  M ^b ) with b values of 0·631, 0·606, 0·655 and 0·650 at 5·0, 8·0, 12·0 and 15·0° C, respectively. The effect of temperature ( T ) and M _D on mean R _R was described by     indicating a Q ₁₀ of 2·27 between 5 and 15° C. Juvenile haddock routine metabolic scope, calculated as the ratio of the mean of highest and lowest deciles of R _R measured in each chamber, significantly decreased with temperature such that the routine scope at 15° C was half that at 5° C. The cost of feeding ( R _SDA) was c . 3% of consumed food energy, a value half that found for larger gadoid juveniles and adults.     

Photosystem II inhibition by moderate light under low temperature in intact leaves of chilling-sensitive and -tolerant plants

Photosystem II (PSII) activity was examsined in leaves of chilling-sensitive cucumber ( Cucumis sativus L.), tomato ( Lycopersicum esculentum L.), and maize ( Zea mays L.), and in chilling-tolerant barley ( Hordeum vulgare L.) illuminated with moderate white light (300 µmol m⁻² s⁻¹) at 4°C using chlorophyll a fluorescence measurements. PSII activity was inhibited in leaves of all the four plants as suggested by the decline in F _v/ F _m, 1/ F _o − 1/ F _m, and F _v/ F _o values. The changes in initial fluorescence level ( F _o), F _v/ F _m, 1/ F _o − /1/ F _m, and F _v/ F _o ratios indicate a stronger PSII inhibition in cucumber, maize and tomato plants. The kinetics of chlorophyll a fluorescence rise showed complex changes in the magnitudes and rise of O-J, J-I, and I-P phases caused by photoinhibition. The selective suppression of the J-I phase of fluorescence rise kinetics provides evidence for weakened electron donation from the oxidizing side, whereas the accumulation of reduced Q_A suggests damage to the acceptor side of PSII. These findings imply that the process of chilling-induced photoinhibition involves damage to more than one site in the PSII complexes. Furthermore, comparative analyses of the decline in F _v/ F _o and photooxidation of P700 explicitly show that the extent of photoinhibitory damage to PSII and photosystem I is similar in leaves of cucumber plants grown at a low irradiance level.     

The Low Affinity Dopamine Binding Site on Tyrosine Hydroxylase: The Role of the N-Terminus and In Situ Regulation of Enzyme Activity

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.     

Mutational analysis of catecholamine binding in tyrosine hydroxylase

Tyrosine hydroxylase (TH) performs the first and rate-limiting step in the synthesis of catecholamines, which feed back to regulate the enzyme by irreversibly binding to a high-affinity site and inhibiting TH activity. Phosphorylation of Ser40 relieves this inhibition by allowing dissociation of catecholamine. We have recently documented the existence of a low-affinity catecholamine binding which is dissociable, is not abolished by phosphorylation, and inhibits TH by competing with the essential cofactor, tetrahydrobiopterin. Here, we have substituted a number of active site residues to determine the structural nature of the low- and high-affinity sites. E332D and Y371F increased the IC(50) of dopamine for the low-affinity site 10-fold and 7 0-fold, respectively, in phosphorylated TH, indicating dramatic reductions in affinity. Only 2-4-fold increases in IC(50) were measured in the nonphosphorylated forms of E332D and Y371F and also in L294A and F300Y. This suggests that while the magnitude of low-affinity site inhibition in wild-type TH remains the same upon TH phosphorylation as previously shown, the active site structure changes to place greater importance on E332 and Y371. Changes to high affinity binding were also measured, including a loss of competition with tetrahydrobiopterin for E332D, A297L, and Y371F and a decreased ability to inhibit catalysis (V(max)) for A297L and Y371F. The common roles of E332 and Y371 indicate that the low- and high-affinity catecholamine binding sites are colocalized in the active site, but due to simultaneous binding, may exist in separate monomers of the TH tetramer.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.     

Inhibition, by 2-Oxo Acids That Accumulate in Maple-Syrup-Urine Disease, of Lactate, Pyruvate, and 3-Hydroxybutyrate Transport Across the Blood-Brain Barrier

Abstract: Data are presented in support of the transport of (-)- d -3-hydroxybutyrate across the blood-brain barrier (BBB) being a carrier-mediated process. The kinetic parameters in 21-day-old pentobarbital-anaesthetized rats were V_max 2.0 μmol.g⁻¹.min⁻¹, K _m 29 m M , and K _D 0.024 ml.g⁻¹.min⁻¹. The value for V_max was the same as that for l -lactate and pyruvate transport in animals of the same age. The transport of all three substrates was sensitive to inhibition by low concentrations of either 2-oxo-3-methylbutanoate or 2-0x0-4-methylpentanoate, the 2-oxo acids that can accumulate in patients with maple-syrup-urine disease. The K _m values for the 2-oxo acids were severalfold lower than the respective K _m values. 2-oxo-3-phenylpropionate was a poor inhibitor. The relative affinities of the various monocarboxylic acids for the transport system of the BBB distinguished it from similar systems described in brain, heart, and liver mitochondria; human erythrocytes; and Ehrlich ascites-tumour cells.     

Transport of Histidine into Synaptosomes of the Rat Central Nervous System

Abstract: Histidine transport into synaptosomes was studied in order to characterize this aspect of histamine synthesis in neurons. Histidine transport was found to be independent of sodium, calcium, and magnesium ions and dependent upon potassium and chloride ions. Histidine transport was also found to be energy dependent, and subcellular fractionation studies suggested it was highly localized to nerve terminals. Kinetic analysis of histidine transport in several brain regions indicated the presence of two uptake sites, a high-affinity site with a K _m of approximately 35 μ M and a low-affinity site with a K _m in the millimolar range. Density of the high-affinity site, as reflected by V_max, correlates well with density of proposed histaminergic innervation. Rate of histidine transport was not altered by prior depolarization of the synaptosomes, indicating that histidine transport probably does not play a regulatory role in histamine synthesis.     

Role of Phe-99 and Trp-196 of sepiapterin reductase from Chlorobium tepidum in the production of L-threo-tetrahydrobiopterin

Sepiapterin reductase from Chlorobium tepidum (cSR) catalyzes the synthesis of a distinct tetrahydrobiopterin (BH4), L -threo-BH4, different from the mammalian enzyme product. The 3-D crystal structure of cSR has revealed that the product configuration is determined solely by the substrate binding mode within the well-conserved catalytic triads. In cSR, the sepiapterin is stacked between two aromatic side chains of Phe-99 and Trp-196 and rotated approximately 180° around the active site from the position in mouse sepiapterin reductase. To confirm their roles in substrate binding, we mutated Phe-99 and/or Trp-196 to alanine (F99A, W196A) by site-directed mutagenesis and comparatively examined substrate binding of the purified proteins by kinetics analysis and differential scanning calorimetry. These mutants had higher K _m values than the wild type. Remarkably, the W196A mutation resulted in a higher K _m increase compared with the F99A mutation. Consistent with the results, the melting temperature ( T _m) in the presence of sepiapterin was lower in the mutant proteins and the worst was W196A. These findings indicate that the two residues are indispensable for substrate binding in cSR, and Trp-196 is more important than Phe-99 for different stereoisomer production.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Purification and properties of an NADPH-dependent dihydroxyacetone reductase from Phycomyces spores

Abstract A glycerol:NADP⁺ 2-oxidoreductase was purified to homogeneity from Phycomyces blakesleeanus sporangiospores. The enzyme had an M _r of 34 000–39 000 and consisted of a single polypeptide. It had a pH optimum between 6–6.5 and a K _m of 3.9 mM for dihydroxyacetone. The reverse reaction had a pH optimum of 9.4 and a K _m for glycerol of more than 2 M. The enzyme was completely specific for NADPH ( K _m= 0.01 mM) or NADP⁺ ( K _m= 0.17 mM) and greatly preferred dihydroxyacetone over glyceraldehyde as substrate. Besides glycerol, l -arabitol and mesoerythritol were also oxidized by the enzyme. It was inhibited by ionic strengths in excess of 100 mM and is probably involved in the synthesis of glycerol during early spore germination.     

Angiotensin-(1–7) through AT2 receptors mediates tyrosine hydroxylase degradation via the ubiquitin–proteasome pathway

Hypothalamic norepinephrine (NE) release regulates arterial pressure by altering sympathetic nervous system activity. Because angiotensin (Ang) (1–7) decreases hypothalamic NE release and this effect may be correlated with a diminished NE synthesis, we hypothesize that Ang-(1–7) down-regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis. We investigated the effect of Ang-(1–7) on centrally TH activity and expression. TH activity was evaluated by the release of tritiated water from ³H- l -tyrosine. TH expression and phosphorylation were determined by western blot. Hypothalami from normotensive or spontaneously hypertensive rats pre-incubated with Ang-(1–7) showed a significant decrease in TH specific activity. Ang-(1–7) caused a decrease in TH phosphorylation at Ser19 and Ser40 residues. The heptapeptide induced a decrease in TH expression that was blocked by an AT₂ receptor antagonist and not by an AT₁ or Mas receptor antagonist, suggesting the involvement of AT₂ receptors. The proteasome inhibitor MG132 blocked the Ang-(1–7)-mediated TH reduction. In addition, Ang-(1–7) increased the amount of TH–ubiquitin complexes, indicating that the Ang-(1–7)-mediated TH degradation involves ubiquitin conjugation prior to proteasome degradation. We conclude that Ang-(1–7) down-regulates TH activity and expression centrally leading to a decrease in the central NE system activity.     

MULTIPLE BINDING SITES OF HUMAN BRAIN MONOAMINE OXIDASE AS INDICATED BY SUBSTRATE COMPETITION

Abstract— Six endogenous substrates of monoamine oxidase (EC 1.4.3.4) (serotonin, l -norepinephrine, dopamine, tyramine, tryptamine and β -phenethylamine) were used separately and in pairs with human brain mitochondrial extracts. Apparent K ₁ values were obtained from experiments in which only 1 of 2 substrates was isotopically labelled, and these values were compared with experimental K _m values. β -Phenethylamine appears to be metabolized at enzyme active sites independent from those which bind serotonin. The substrate l -norepinephrine competes with serotonin for an enzyme site, but also may be catalysed at an additional site which is independent of serotonin binding. Experiments in which [¹⁴C]tryptamine was combined with [³E]serotonin indicated that tryptamine is a much more potent inhibitor of serotonin oxidation than was predicted from K _m values. It is suggested that the competition among substrates of MA0 which is observed in uitro may have relevance to in uiuo mechanisms for control of biogenic amine concentrations.     

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high- K _m activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low- K _m activity was also present in the cytosol (<20%). The low- K _m activity in the homogenate was only 10–15% of the total activity with acetaldehyde as the substrate. Two K _m values were obtained with both acetaldehyde (0.2 and 2000 μ m ) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 μ m ), and one K _m value with succinate semialdehyde (5 μ m ). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low- K _m activity with acetaldehyde and DOPAL, but not the high- K _m activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg²⁺ and Ca²⁺ in phosphate buffer. The low- and high- K _m activities with acetaldehyde showed different pH optima in pyrophosphate buffer.     

Phorbol Ester Administration Transiently Increases Aromatic l-Amino Acid Decarboxylase Activity of the Mouse Striatum and Midbrain

Abstract: Aromatic l -amino acid decarboxylase (AAAD) is required for the synthesis of catecholamines, serotonin, and the trace amines. We found that the protein kinase C activator phorbol 12-myristate 13-acetate administered intracerebroventricularly transiently increased AAAD activity by 30–50% over control values within ∼30 min in the striatum and midbrain of the mouse. The enzyme increase was manifested as an apparent increase of V _max with little change of K _m for either l -3,4-dihydroxyphenylalanine or pyridoxal phosphate. Chelerythrine, a protein kinase C inhibitor, prevented the phorbol ester-induced increase of AAAD. Moreover, okadaic acid, a serine/threonine-selective protein phosphatase 1 and 2A inhibitor, also increased AAAD activity in the mouse striatum and midbrain. Taken together, these observations suggest that protein kinase C-mediated pathways modulate AAAD activity in vivo.     

Energy density of anchovy Engraulis encrasicolus L. in the Adriatic Sea

European anchovy Engraulis encrasicolus , with total lengths ranging from 40·0 to 132·5 mm, were sampled during October 2002 and May 2003 in the northern Adriatic Sea in order to estimate their energy densities ( E _D). A highly significant ( P  < 0·001) relationship between E _D(y)(J g⁻¹wet mass) and per cent dry mass ( x ) was found: y  = 321 x  − 3316·9 ( n  = 161, r ² = 0·82).