Relationship between tryptophan metabolism and the state of melaninogensis]

In studying the excretion of tryptophan and the activity of tryptophan pyrrholase of the liver in black and albino rabbits it was revealed that the kinurenine, kinurenic and xanthurenic acids, 3-hydroxykinurenin, anthranylic acid and 5-hydroxyindolylactic acid levels in the urine collected before the L-tryptophane loading differed in these animals. No 3-hydroxyanthranylic acid was found in the initial urine of these rabbits. A sharp intensification of excretion of all the tryptophane metabolites studied followed the administration of L-tryptophane to albino rabbits, and a harper elevation of kinurenic and xanthurenic acid excretion--to black rabbits. In comparison with black rabbits, the tryptophan pyrrholase of the liver of albino rabbits reached to the L-tryptophan administration more intensively.     

Possible role of octopamine and tyramine in the antihypertensive and antidepressant effects of tyrosine

The administration of a dose of 200 mg/kg of tyrosine (as either the free amino acid or the ethyl ester) increased the 24-hour excretion of p-hydroxyphenethyleneglycol (p-HPG) and p-hydroxyphenylethanol, metabolites of octopamine and tyramine, by 147 and 50%, respectively. One hour after this dose of tyrosine, brain levels of p-HPG and p-hydroxyphenylacetic acid (p-HPA), another metabolite of tyramine, were increased by 82 and 196%, respectively. Pretreatment with Ro4-4602, a peripheral decarboxylase inhibitor, reduced by 50% the tyrosine-induced increases in brain p-HPA levels, suggesting that tyramine was partially formed in the brain parenchyma. Tyrosine caused only slight, but non-significant increases in brain levels of catecholamine metabolites. These results suggest that tyrosine-induced increases in the production of tyramine and octopamine in brain may account for some of the effects of tyrosine, such as its antihypertensive and reported antidepressant properties.     

Human TRP-1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (≤0.8 μCi/ml of ³H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.     

Tyrosine and methionine metabolism in various states of melaninogenesis

Excretion with urine of tyrosine and methionine metabolites as well as the activities of enzymes involved in their metabolism are correlated with the state and type of melanin synthesized in the skin. The response of tyrosine aminotransferase to melaninogenesis induction was more pronounced in animals with predominant pheomelaninogenesis, especially after tyrosine load, while that to dopachrome oxidoreductase--in animals with predominant eumelaninogenesis and after methionine load. Glutathione reductase and cystathionine-beta-synthase responded more vigorously to methionine injections, which was especially well pronounced in animals with prominent pheomelaninogenesis and in albino animals. The metabolic "block" in melanine synthesis in albino animals seems to be observed after the 5-S-cysteinyl-DOPA synthesis, whereas the initial steps of melaninogenesis in these animals are identical to pheomelanine synthesis reactions.     

Metabolic Influences on Tyrosine Excretion in Bacillus subtilis

The biosynthetic pathway for tyrosine synthesis is regulated both by repression of enzyme synthesis and by feedback inhibition of enzyme activity in Bacillus subtilis. Nevertheless, wild-type cells produce significantly more tyrosine than is required for protein synthesis, and part of this is excreted into the medium. Alteration of nutritional and other environmental conditions of cultivation strongly influenced the amount of tyrosine excretion. The excretion of tyrosine by wild-type cells was compared with that of a regulatory mutant having a feedback-insensitive prephenate dehydrogenase. Tyrosine excretion varied directly with the in vitro activity of prephenate dehydrogenase and inversely with temperature in the two strains. The regulatory mutant excreted five times as much tyrosine as the wild type at all growth temperatures examined. The carbon source used for growth significantly influenced the level of tyrosine excretion. The specific activity of prephenate dehydrogenase was also affected by the carbon source. Incorporation studies with isotopic tyrosine and fluorometric determinations of tyrosine concentrations extractable in hot water were used to measure operationally the tyrosine pools in the mutant and wild-type strains. The effects of various environmental conditions on the synthesis and excretion of tyrosine led to the conclusion that metabolic controls governing end product contrations exist which are completely independent of regulation by feedback inhibition and repression.     

Effects of two different loading doses of L-tryptophan on the urinary excretion of tryptophan metabolites in rats, mice and guinea pigs. Correlation with the enzyme activities

The effect of two different loading doses of L-tryptophan (0.5 and 1.0 g/Kg b.w.) on excretion of tryptophan metabolites and the relation to the enzyme activities were studied in rats, mice and guinea pigs. In rats there is no ratio between the dosage used and the levels of the metabolites excreted. Doubling the amount of tryptophan administered, a 5-fold increase in the elimination of the metabolites along the kynurenine pathway is obtained. The 1.0 g/Kg load provides a more complete pattern of the metabolites than with the 0.5 g/Kg b.w. load. Kynurenic acid, kynurenine and xanthurenic acid are the chief metabolites excreted. In mice, the urinary excretion of the metabolites is very low with both loads. In guinea pigs, xanthurenic acid is excreted in the highest amount and kynurenic acid and kynurenine also constitute the large fractions with both loadings. The load of 0.5 g/Kg b.w. is preferable to that of 1.0 g/Kg b.w. for not causing B6-deficiency. Liver tryptophan pyrrolase exists in two forms in rats, while in mice and in guinea pigs it is present only as holoenzyme. This enzyme is more active in rats than in the other two species of animals. Kynureninase activity is lower in guinea pigs, but it apparently correlated to the low levels of excretion of the metabolites following this step. Kynurenine aminotransferase is very active in rats and in mice, while it is apparently depressed in guinea pigs, in contrast with the high excretion of xanthurenic and kynurenic acids, that puts in evidence a B6-deficiency. The excretion of tryptophan metabolites and enzyme activities are better correlated in rats.     

Effect of cofactor depletion on liver tyrosine aminotransferase expression.

Hepatic tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) was partially purified from pyridoxine depleted and control rats and subsequently resolved by electrophoresis on polyacrylamide gels. Enzyme activity was detected histochemically in situ on the gel. Six enzymatically active forms were detected. Cofactor depletion effected further resolution of the enzyme into seven active forms as revealed by the bifurcation of the major active peak.     

Differences in properties between aromatic amino acid: Aromatic keto acid aminotransferases and aromatic amino acid: α-ketoglutarate aminotransferases

Homogenates of rat liver transaminate phenylpyruvate (PP), as well as α-ketoglutarate (α-KG), in the presence of L-tyrosine, 3,4-dihydroxyphenylalanine (L-DOPA) or L-tryptophan. Aminotransferase activity with phenylpyruvate and DOPA, but not with tyrosine, was inhibited by excess phenylpyruvate. Tyrosine and DOPA aminotransferase activities with phenylpyruvate were more heat stable than the corresponding activities with α-ketoglutarate. Aminotransferase activities with phenylpyruvate were not significantly induced following intraperitoneal injections of cortisol, glucagon or serotonin, compared with a 3 to 7-fold increase in the aminotransferase activities with α-ketoglutarate. Tyrosine:phenylpyruvate aminotransferase activity rose 40% at night, compared with a 300% increase in tyrosine:α-ketoglutarate aminotransferase activity. The results suggest that aminotransferases catalysing transfers between aromatic keto acids and aromatic amino acids are separate enzymes from those utilizing α-ketoglutarate as the acceptor keto acid.     

Vitiligo,Psoralen, and Melanogenesis: Some Observations and Understanding

Since the etiology of vitiligo is still unknown, we searched for some abnormal biochemical parameters, if any, in subjects with vitiligo. Higher urinary excretion of indole metabolites in vitiliginous patients have been noted, in association with higher dioxygenase, superoxide dismutase, and tyrosine aminotransferase activity in their serum. Similar results have also been found in an animal model, Bufo melanostictus, during induced tyrosinase inhibition. Treatment with psoralen can reverse the parameters, except tyrosine aminotransferase, to a normal level. Although psoralens are not the magic bullet for the therapy of vitiligo, they are still being used as a chemotherapeutic agent against vitiligo on a major scale to date. Tryptophan was found to participate in the pathway of melanogenesis, as a precursor as well as a positive regulator of tyrosinase. Its behavior in this regard is much more similar to the conventional substrates tyrosine and dopa (dihydroxyphenylalanine). In consideration of combined participation of tyrosine and tryptophan in the synthesis of melanin and its breakdown, the possible influence of different enzymatic reactions, like mono-oxygenase, dioxygenase, and deamination, has been suggested.     

Alterations in Retinal Tyrosine and Dopamine Levels in Rats Consuming Protein or Tyrosine-Supplemented Diets

The ad libitum ingestion of casein diets varying in protein content altered serum and retinal levels of tyrosine. The serum tyrosine level rose when protein ingestion was increased from 6 to 24% casein. In rats consuming high-protein diets (40% casein), no further increase in serum tyrosine level occurred, although the levels of other large neutral amino acids, which compete with tyrosine for retinal uptake, continued to rise. The activity of the liver enzyme tyrosine aminotransferase varied directly with the percentage of protein in the diet and may partially explain the failure of chronic high-protein feeding to increase serum tyrosine levels. The retinal tyrosine concentration was significantly correlated with the serum tyrosine level and with the serum tyrosine ratio at all levels of protein intake. Retinal 3,4-dihydroxyphenylalanine synthesis and dopamine (DA) level varied in parallel with the level of the precursor, tyrosine. Addition of pure L-tyrosine (1, 2, or 4%) to normal protein diets resulted in a stepwise increase in serum and retinal tyrosine levels and retinal DA turnover. Alterations of retinal tyrosine level as a result of change in amount of dietary protein or by its addition to the normal diet can influence retinal DA synthesis and release.     

Biotransformation of 3-(3′,5′ -Dichlorophenyl)-5,5-dimethyl oxazolidine-2,4-dione

Using 3-(3′,5′-dichlorophenyl)-5,5-dimethyloxazolidine-2,4-dione labeled with ¹⁴C or ³H, absorption, excretion, and tissue distribution in male Wistar rats were studied, and metabolites excreted were identified. At the dosage rates of 100, 300, 1000 and 3000 mg/kg, the maximum excretion of orally administered radioactivity occurred within 24 hr. Increase in the dosage rate was paralleled by decrease in the proportion of urinary elimination. Essentially all the radioactivity was excreted in 2 weeks. DDOD level was generally low in most tissues. Adipose tissue contained higher radioactivity compared with others. Most of the urinary metabolites identified were characterized by hydroxylation at the 4′ position of the benzene ring moiety, and hydrolytic or oxidative modification of the oxazolidine ring portion.     

The biotransformation and urinary excretion of dexamethasone in equine male castrates

The pro-drugs of dexamethasone, a potent glucocorticoid, are frequently used as anti-inflammatory steroids in equine veterinary practice. In the present study the biotransformation and urinary excretion of tritium labelled dexamethasone were investigated in cross-bred castrated male horses after therapeutic doses. Between 40-50% of the administered radioactivity was excreted in the urine within 24 h; a further 10% being excreted over the next 3 days. The urinary radioactivity was largely excreted in the unconjugated steroid fraction. In the first 24 h urine sample, 26-36% of the total dose was recovered in the unconjugated fraction, 8-13% in the conjugated fraction and about 5% was unextractable from the urine. The metabolites identified by microchemical transformations and thin-layer chromatography were unchanged dexamethasone, 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone together accounting for approx 60% of the urinary activity. About 25% of the urinary radioactivity associated with polar metabolites still remains unidentified.     

Stabilization and purification of tyrosine aminotransferase from rat liver

Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation by prevented. The enzyme was stabilized by pyridoxal 5'-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-glutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65 degrees C) followed by precipitation from 0.20 M sucrose at pH 5.0; and small-scale chromatography on DEAE-cellulose, hydroxyapatite and CM-Sephadex C50 at pH 6.0. These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity.     

Stabilization of rat liver tyrosine aminotransferase by tetracycline.

Rat liver tyrosine aminotransferase was purified 200-fold and an antiserum raised against it in rabbits. 2. Hepatic tyrosine aminotransferase activity was increased fourfold by tyrosine, twofold by tetracycline, 2.5-fold by cortisone 21-acetate and ninefold by a combination of tyrosine and cortisol administered intraperitoneally to rats. 3. Radioimmunoassay with 14C-labelled tyrosine aminotransferase, in conjunction with rabbit antiserum against the enzyme, revealed that cortisol stimulates the synthesis of the enzyme de novo, but that tetracycline has no such effect. 4. Incubation of rat liver homogenates with purified tyrosine aminotransferase in vitro leads to a rapid inactivation of the enzyme, which tetracycline partially inhibits. 5. The inactivation is brought about by intact lysosomes, and the addition of 10mM-cysteine increases the rate of enzyme inactivation, which is further markedly increased by 10mM-Mg2+ and 10mM-ATP. Here again tetracycline partially inhibits the decay rate, leading to the inference that the increase of tyrosine aminotransferase activity in vivo by tetracycline is brought about by the latter inhibiting the lysosomal catheptic action.     

Comparative proteomics in alkaptonuria provides insights into inflammation and oxidative stress

Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism associated with a defective catabolism of phenylalanine and tyrosine leading to increased systemic levels of homogentisic acid (HGA). Excess HGA is partly excreted in the urine, partly accumulated within the body and deposited onto connective tissues under the form of an ochronotic pigment, leading to a range of clinical manifestations. No clear genotype/phenotype correlation was found in AKU, and today there is the urgent need to identify biomarkers able to monitor AKU progression and evaluate response to treatment. With this aim, we provided the first proteomic study on serum and plasma samples from alkaptonuric individuals showing pathological SAA, CRP and Advanced Oxidation Protein Products (AOPP) levels. Interesting similarities with proteomic studies on other rheumatic diseases were highlighted together with proteome alterations supporting the existence of oxidative stress and inflammation in AKU. Potential candidate biomarkers to assess disease severity, monitor disease progression and evaluate response to treatment were identified as well.     

Concanavalin A alters the turnover rate of tyrosine aminotransferase in cultured hepatoma cells

Concanavalin A added to monolayer cultures of Reuber H-35 hepatoma cells caused a rapid inactivation of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, E.C. 2.6.1.5) and loss of reactivity with antibody against the native, dimeric enzyme. Analysis of treated cells with an antibody raised against carboxymethylated, denatured enzyme showed that the inactivated enzyme was reactive with this reagent, which does not react with the native enzyme. Subsequent addition of alpha-methyl-D-mannopyranoside to remove concanavalin A restored both enzyme activity and reactivity to antibody against native enzyme. After long-term treatment with concanavalin A, the restored enzyme levels were significantly higher than in controls treated with the sugar but not the lectin. Analysis of the turnover of the enzyme by two methods revealed that the rate of its degradation is reduced about 2-fold in concanavalin A-treated cells. Treatment with H-35 cells with concanavalin A thus effects an alteration in conformation of tyrosine aminotransferase, rendering it somewhat less sensitive to intracellular degradation.     

Pretranslational control of tyrosine aminotransferase synthesis by 8-bromo-cyclic AMP in H-4 rat hepatoma cells

The H-4 rat hepatoma cell line grown in tissue culture was used as a model system to investigate the action of cAMP in tyrosine aminotransferase induction. An immunoprecipitation technique was used to quantitate the amount and the rate of synthesis of tyrosine aminotransferase; the level of mRNA coding for tyrosine aminotransferase was determined by in vitro translation of poly(A)+ RNA isolated from hepatoma cells. Our results demonstrated that 8-bromo-cAMP gave time-dependent and proportionate increases in the tyrosine aminotransferase activity, the amount of immunoprecipitable tyrosine aminotransferase, the rate of synthesis of tyrosine aminotransferase, and the level of mRNATAT in H-4 hepatoma cells. The time course of increase in mRNATAT preceded the increase in synthesis of tyrosine aminotransferase and was dependent on the continuous production of poly(A)+ RNA. Pretreatment of the cells with cordycepin completely abolished the 8-bromo-cAMP-evoked increase in mRNATAT activity. These results provided evidence that the primary action of cAMP in tyrosine aminotransferase induction is the increase of functional mRNATAT and that this increase can completely account for the increase in tyrosine aminotransferase activity.     

Protection of tyrosine aminotransferase against proteolytic digestion by nucleotide derivatives

The abilities of several nucleotides to protect tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) against proteolytic inactivation in vitro have been examined as part of an ongoing investigation of the role of cyclic GMP in the intracellular degradation of the hepatic enzyme. Although neither cyclic GMP nor cyclic AMP was found to exert such a protective effect, certain nucleotide analogs were observed to inhibit the inactivation of tyrosine aminotransferase by trypsin and chymotrypsin. The nucleotides which conferred the strongest protection were the dibutyryl derivatives of cyclic GMP and cyclic AMP. This phenomenon appears to require a purine nucleotide with hydrophobic substituent(s), while the cyclic phosphate is not essential. The nucleotides probably act by direct interaction with tyrosine aminotransferase as indicated by changes in kinetic properties and heat stability of the enzyme and by their failure to inhibit trypsin when other protein substrates, including another aminotransferase, were used. Dibutyryl cyclic AMP was shown to block the appearance of a characteristic 43 kDa tryptic cleavage product of tyrosine aminotransferase but not the conversion of the native 54 kDa form to a size of 50 kDa. Arguments are presented against the involvement of the protective effect in the actions of dibutyryl cyclic nucleotides on tyrosine aminotransferase in cells.