Transformation of difluorinated phenols by Penicillium frequentans Bi 7/2

The Penicillium frequentans strain Bi 7/2, using phenol as a sole source of carbon and energy,transformed the fluorinated phenols 2,3-, 2,4-, 2,5-and 3,4-difluorophenol rapidly. After growth on phenol, resting mycelia of the fungus converted the difluorophenols completely at an initial concentration of 0.5 mM within 6 hours. The corresponding difluorinated catechols were found to be intermediates of all difluorophenols investigated. A relatively unspecific phenol hydroxylase catalyzed this hydroxylation step and showed activities towards all difluorophenols tested. One difluorocatechol was formed from each difluorophenol substituted with fluorine in the ortho-position, whereas two catechols were formed from 3,4-difluorophenol, due to its two vacant ortho-positions. A partial defluorination (50-77%) was observed in all cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

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 in the cerebral ganglia of the American cockroach (Periplaneta americana L.): an immunohistochemical study

We have investigated the distribution of tyrosine-hydroxylase-like immunoreactivity in the cerebral ganglia of the American cockroach, Periplaneta americana. Groups of tyrosine-hydroxylase-immunoreactive cell bodies occur in various parts of the three regions of the cerebral ganglia. In the protocerebrum, single large neurons or small groups of neurons are located in the lateral neuropil, adjacent to the calyces, and in the dorsal portion of the pars intercerebralis. Small scattered cell bodies are found in the outer layers of the optic lobe, and clusters of larger cell bodies can be found in the deutocerebrum, medial and lateral to the antennal glomeruli. Thick bundles of tyrosine-hydroxylase-positive nerve fibers traverse the neuropil in the proto- and deutocerebrum and innervate the glomerular and the nonglomerular neuropil with fine varicose terminals. Dense terminal patterns are present in the medulla and lobula of the optic lobe, the pars intercerebralis, the medial tritocerebrum, and the area surrounding the antennal glomeruli, the central body and the mushroom bodies. The pattern of tyrosine-hydroxylase-like immunoreactivity is similar to that previously described for catecholaminergic neurons, but it is distinctly different from the distribution of histaminergic and serotonergic neurons.     

Methylfolate modulates potassium evoked neuro-secretion: Evidence for a role at the pteridine cofactor level of tyrosine 3-hydroxylase

We have previously shown that 5-methyltetrahydrofolate influences neuro-secretion. The present study more precisely characterises the processes involved and considers one probable site of action. Focusing on the tyrosine-noradrenalin axis in cerebellum we showed 5-methyltetrahydrofolate causes a significant reduction in the apparent K⁺ evoked secretion of noradrenalin to only 12.9% of control release. Evidence supports the idea that this could actually be due to increased synthesis leading to; depletion of reserves, possibly through leakage, exocytotic inhibition via activation of presynaptic receptors or end product inhibition by noradrenalin at the pteridine cofactor level of tyrosine hydroxylase: a) concomitant decreased measurement of perfusate and intracellular tyrosine with released noradrenalin following 5-methyltetrahydrofolate treatment supports the idea of increased transmitter turn over; b) kinetic studies indicate that at saturating concentrations of tyrosine and in the presence of an inhibitor of L-DOPA decarboxylase, 5-methyltetrahydrofolate partially duplicates the rate limiting behaviour of a synthetic pteridine cofactor — DL,2-amino-4-hydroxy-6,7,dimethyltetrahydropteridine. We debate whether, in vivo, CSF 5-methyltetrahydrofolate might interact at the tetrahydrobiopterin cofactor level of tyrosine hydroxylase and other aromatic amino-acid hydroxylases.     

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver microsomes which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP). α-Naphthoflavone (ANF) selectively inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (M_r = 56 000). However, HCB did induce a polypeptide with M_r = 53 000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the ‘mixed-type’ induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.     

Morphine and Cocaine Exert Common Chronic Actions on Tyrosine Hydroxylase in Dopaminergic Brain Reward Regions

We studied levels of tyrosine hydroxylase immunoreactivity and phosphorylation state in the ventral tegmental area (VTA) and nucleus accumbens (NAc) in an effort to understand better the mechanisms by which these brain reward regions are influenced by opiates and cocaine. In the VTA, chronic, but not acute, administration of either morphine or cocaine increased levels of tyrosine hydroxylase immunoreactivity by 30-40%, with no change observed in the relative phosphorylation state of the enzyme. In the NAc, chronic, but not acute, morphine and cocaine treatments decreased the phosphorylation state of tyrosine hydroxylase, without a change in its total amount. In contrast, morphine and cocaine did not regulate tyrosine hydroxylase in the substantia nigra or caudate/putamen, brain regions generally not implicated in drug reward. Morphine and cocaine regulation of tyrosine hydroxylase could represent part of a common biochemical basis of morphine and cocaine addiction and craving.     

Vanillic acid metabolism by selected soft-rot,brown-rot,and white-rot fungi

Metabolism of vanillic acid, a product of lignin degradation, has been studied in selected representatives of soft-rot, brown-rot and white-rot fungi. All of the brown-and white-rot species examined decarboxylated vanillate to methoxyhydroquinone oxidatively. Mycelium extracts of all these fungi, except Pleurotus ostreatus contained high levels of an NAD(P)H-dependent vanillate hydroxylase. P. ostreatus also released ¹⁴CO₂ from ¹⁴COOH-vanillate but by a different mechanism possibly involving phenoloxidases. Most of these fungi also contained a dioxygenase which catalysed the intra-diol cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to form maleylacetate. No 3-O-demethylase activity was detected, and data indicate that in some of the fungi examined cleavage of the aromatic ring occurs without prior removal of the methoxyl group. None of the soft-rot fungi tested contained vanillate hydroxylase or hydroxyquinol 1,2-dioxygenase, but very low levels of protocatechuate 3,4-dioxygenase were detected in mycelium extracts. Vanillate catabolism among members of this group occurs via a different route which may involve ring demethylation although no 3-O-demethylase activity was detected in this study. The enzyme NAD(P)H-quinone oxidoreductase was demonstrated to exist in all the studied groups of fungi.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.