The influence of glucose, other monosaccharides, and ascorbic acid on tyrosine hydroxylase activity of rat striatal synaptosomes.

We investigated the action of glucose, other monosaccharides, and ascorbic acid on the activity of tyrosine hydroxylase in rat striatal synaptosomes. We found that glucose at 0.2 mM maximally activated enzyme activity by as much as 100 percent and caused half-maximal activation at 0.036 mM. Mannose, fructose and galactose also stimulated tyrosine hydroxylase activity, half-maximal activation occurring at 0.036, 8, and 50 mM, respectively; arabinose was inactive up to 100 mM. Ascorbic acid did not stimulate enzyme activity at 0.1 and 1 mM, and at 10 mM was inhibitory.The activating effect of glucose on tyrosine hydroxylase activity was blocked by 2-deoxyglucose and by glucosamine. We interpret the action of glucose to be dependent upon its metabolism and to be indirect, probably due to the maintenance of the cofactor in the reduced form in the synaptosomes.     

Regulation of tyrosine hydroxylase activity in the rat hypothalamus with gamma-aminobutyric acid

It is established that GABA interacts with tyrosine hydroxylase through the allosteric site which is not identical to sites of tyrosine, DOPA, pterin cofactor, dopamine binding. This interaction is very significant in the GABA influence on the regulation of the tyrosine hydroxylase activity by presynaptic receptors. GABA is supposed to be able to cause dissociation of oligomeric forms of tyrosine hydroxylase.     

Regulatory mechanism of tyrosine hydroxylase activity

Activity of tyrosine hydroxylase is regulated by feedback inhibition and inactivation by catecholamines, and activation by protein phosphorylation. In this article, reaction mechanisms for the conversion of tyrosine hydroxylase to an inactive/stable form by catecholamines, and activation of tyrosine hydroxylase by phosphorylation at Ser-40 are discussed. Inactivation may be induced by sub-stoichiometric amounts of catecholamines, and activation by phosphorylation of Ser-40 may require phosphorylation of three or all four subunits of a tyrosine hydroxylase molecule. Cooperative phosphorylation at Ser-40 in the subunits is also discussed.     

Effects of polyamines on tyrosine hydroxylase activity in adrenals

The ability of polyamines (putrescine, spermidine, and spermine) to modify tyrosine hydroxylase (TH) activity was examined in crude or purified enzyme preparation and in adrenal tissue slices. Polyamines showed biphasic effects on TH activity in vitro at physiological pH 7.0, with an inhibitory effect at low concentrations (<1 mM) and a stimulatory effect at high concentrations. The degree of both inhibition and stimulation produced by polyamines at low and high concentrations, respectively, were proportional to the number of the amino group in the polyamines (putrescine < spermidine < spermine). The degree of inhibition by polyamines was much greater with purified enzyme than with crude enzyme preparations. Tyrosine hydroxylation in situ in adrenal tissue slices was stimulated by polyamines without inhibition at any concentrations tested. This evidence suggests that TH molecules in vivo could interact with polyamines or polyamine-like substances which inhibit the TH activity at physiological concentrations less than 1 mM.     

Studies on the mutagenic activity of ascorbic acid in vitro and in vivo

In vitro data are presented to show that ascorbic acid does not have intrinsic mutagenicity towards strain TA100 of S. typhimurium if deionized water is used to prepare the incubation medium. The addition of Cu2+ ions to the bacterial medium that contains ascorbic acid, or the use of tap water and ascorbic acid alone, causes a mutagenic and cytotoxic response that is blocked by EDTA. Additional in vitro data demonstrate that hydrogen peroxide is mutagenic to S. typhimurium strain TA100 and it is suggested that ascorbic acid may be mutagenic and cytotoxic through the generation of hydrogen peroxide. In vivo studies using a sensitive intrahepatic host-mediated mutagenicity assay indicate that ascorbic acid is not genotoxic in guinea pigs even when the dietary intake of vitamin C is above the level required for tissue saturation (5000 mg/kg body weight/day).     

Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine

The effects of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and kidney carnitine levels were not affected by the deficiency, but scorbutic animals had 50% less carnitine in heart and skeletal muscle than control animals. Labeled carnitine precursors, 6-N-tri-methyl-L-lysine and 4-N-trimethylaminobutyrate, both of which require ascorbate for their enzymatic hydroxylation, were injected into the vena cava of control, pair-fed and scorbutic animals. The distribution of isotope in compounds present in the liver and kidney after 1 h was determined. The uptake of trimethyllysine by the liver was less than 2% in 1 h, while the kidney took up approx. 20% of the 14C. Control and pair-fed animals converted trimethyllysine to kidney trimethylaminobutyrate 8--10 times as well as did scorbutic animals. Trimethylaminobutyrate hydroxylase, present in the liver but almost absent from the kidney, converted nearly all of substrate taken up by the liver to carnitine in both the scorbutic and control animals.     

Neuroleptics-induced changes of tyrosine hydroxylase activity in rat striatum in vitro and in vivo.

The potency of haloperidol and chlorpromazine, but not clozapine, for increasing homovanillic acid and activating tyrosine hydroxylase in the striatum was significantly weakened after the repeated administration in rats. These findings suggest that clozapine could supply enough dopamine to surmount the blockade of dopamine receptors in the striatum even after the repeated administration. This property of clozapine seems to be the cause of low incidence of extrapyramidal side effects in clinical use.     

Brain tyrosine hydroxylase activity in behavior-selected silver foxes

In two groups of silver foxes--i.e. selected by the domestic type of behaviour and aggressive ones--studies have been made on the activity of the key enzyme in biosynthesis of catecholamines--i.e. tyrosine hydroxylase from the brain. Domesticated animals exhibited higher enzymic activity in the locus coeruleus, hypothalamus and cortex. Animals from both groups did not differ with respect to the level of tyrosine hydroxylase activity in the corpus striatum. The enzymic reactions of homogenates from locus coeruleus region of the brain in both groups of animals, as well as homogenates from the corpus striatum of the brain of aggressive animals exhibited low and approximately equal values of Michaelis constant for tyrosine. The value of KM was 3 times higher in the hypothalamus in both groups of foxes and in the corpus striatum of tame animals. Presumably, selection of silver foxes for the domestic type of behaviour resulted in the increase of biosynthesis of catecholamines in the brain due to the increase in the number of enzyme molecules. The increase in the activity of tyrosine hydroxylase in noradrenaline system of the brain may be associated with changes in the behavioural pattern of animals resulting from selection.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Ciliary neurotrophic factor stimulates tyrosine hydroxylase activity

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in norepinephrine synthesis, and its expression and activity are regulated by many factors in sympathetic neurons. Cytokines that act through gp130, such as ciliary neurotrophic factor (CNTF) decrease norepinephrine production in sympathetic neurons by suppressing TH mRNA and stimulating degradation of TH protein, leading to the loss of enzyme. Their effect on the activity of TH is unclear, but recent in vivo observations suggest that cytokines may stimulate TH activity. We investigated this issue by quantifying TH protein levels and activity in cultured sympathetic neurons. We also examined the state of TH phosphorylation on serine 31 and 40, sites known to affect TH activity and degradation. We found that CNTF, acting through gp130, stimulated the rate of l-3,4-dihydroxyphenylalanine production while at the same time decreasing TH enzyme levels, thereby increasing the specific activity of the enzyme. We also found that phosphorylation of TH on Ser31 was increased, and phosphorylation on Ser40 was decreased, after four days of CNTF exposure. Our data are consistent with previous findings that Ser31 phosphorylation stimulates TH activity, whereas Ser40 phosphorylation can target TH for proteasomal degradation.     

Genetic determination of striatal tyrosine hydroxylase activity in mice

An additive major gene effect is described for tyrosine hydroxylase activity in mouse corpus striatum (CS). Quantitative genetic analysis indicated the presence of a segregating Mendelian factor with robust additive effect in F₂ generations derived from crossing two highly inbred mouse strains, C57BL/6ByJ and BALB/ cJ, with intermediate (INT) and high (HI) TH activity in CS. Significant positive correlation was found between striatal and mesencephalic TH activity in the segregating generations, raising the possiblity that a common single gene may express its effect through pleiotropy or linkage. Genetic preparations taking advantage of the major gene effect should serve well as animal models of DA-mediated neuropsychiatric disorders.     

Pb-induced alterations in tyrosine hydroxylase activity in rat brain

It was concluded that cytochrome oxidase was a strange enzyme for three reasons. (1) The thermodynamic flux-force relationship of this enzyme was inverse in some conditions: flux decreased when force increased. (2) The flux-force relationship was not unique and depended on the way in which the thermodynamic span of cytochrome oxidase was changed. (3) The regulation of cytochrome oxidase was different in the same conditions when different external parameters (energy demand, oxygen concentration) were changed.It was also shown that the flux control coefficient of cytochrome oxidase, small at saturating oxygen concentration, increases when oxygen pressure diminishes, approaching unity at very low oxygen concentrations. (Mol Cell Biochem 174: 137–141, 1997)     

Effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice

The effect of exogenous ascorbic acid intake on biosynthesis of ascorbic acid in mice has been studied. After the mice were on diets containing added ascorbic acid for two months, the activities of ascorbic acid synthesizing enzymes in the mouse liver homogenates were measured using L-gulono-gamma-lactone as a substrate. Exogenous ascorbic acid intake (0.5, 1 or 5% in the diet) was able to increase the concentration of ascorbic acid in the blood and to decrease the activities of ascorbic acid synthesizing enzymes in mouse liver. The results suggest that ascorbic acid synthesis was controlled by local regulatory mechanism or by the concentration of ascorbic acid in the hepatic portal blood. Ingestion of dietary erythorbic acid, a stereoisomer of ascorbic acid, had no effect on the activities of ascorbic acid synthesizing enzymes.     

Allosteric effect of tetrahydrobiopterin cofactors on tyrosine hydroxylase activity.

Allostery of tyrosine hydroxylase was found by kinetical studies of partially purified tyrosine hydroxylase from clonal rat pheochromocytoma PC12h cells. Positive cooperativity toward the cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6R)BH4] and (6S)-L-erythro-5,6,7,8-tetrahydrobiopterin [(6S)BH4], was observed. It is indicated that biopterin might be the regulatory factor of the enzyme polymers, which changes the affinity for the cofactor itself. Moreover, the stereochemical structure of (6R)BH4, the naturally-occurring cofactor, took an important role on the kinetical properties of the enzyme in concern with L-tyrosine.