Junko Naito

Skin l-tryptophan-2,3-dioxygenase and rat hair growth

We have identified a new enzyme, skin l‐tryptophan‐2,3‐dioxygenase (skin TDO), that catalyzes the degradation of l‐tryptophan into formylkynurenine in rats. The rate of this degradation peaks in all rats at 5 to 6 weeks after birth, and also, among rats depilated at 8 weeks old, at 10 to 11 weeks after birth. We have also observed that the properties of this enzyme are closer to those of hepatic l‐tryptophan‐2,3‐dioxygenase (hepatic TDO) than to indoleamine 2,3‐dioxygenase. Although an intraperitoneal injection of l‐tryptophan increased the activity of skin TDO to approximately 2.2 times greater than control values, an intraperitoneal injection of hydrocortisone and α‐methyl‐dl‐tryptophan, both compounds known to affect hepatic TDO activity, had no effect on skin TDO activity. The molecular weight of skin TDO was estimated to be 16.0 kDa, which is close to the molecular weight of hepatic TDO, yet a much larger molecule than indoleamine‐2,3‐dioxygenase. Increased hair growth rates paralleled increased levels of skin TDO activity in 5‐ to 6‐week‐old rats, and marked increases in the activity of skin TDO occurred 2 or 3 weeks after depilation. Enzyme activity was also greatest 2 days before the time of maximum hair root length. Therefore, skin TDO may play an important role in the initiation or suppression of rat hair growth.

Determination of kynurenine in serum by high-performance liquid chromatography after enzymatic conversion to 3-hydroxykynurenine

A method for the determination of the physiological level of kynurenine in human serum based upon conversion of kynurenine to 3-hydroxykynurenine by enzymatic reaction with the mitochondrial fraction and NADPH and analysis by reversed-phase high-performance liquid chromatography with electrochemical detection is described. Tryptophan gave no interference. For one analysis, 0.2 ml of serum was sufficient, compared with the large volume (5.0 ml) required for other methods.

Tryptophan 2,3-dioxygenase activity in rat skin

We have found an enzyme system that catalyzes the conversion of L-tryptophan to L-kynurenine, presumably via L-formylkynurenine, in soluble and insoluble fractions of rat skin. The enzymatic activity was stimulated by hematin, ascorbate, and catalase, but not by methylene blue. Highest activity was located in the skin of the dorsal posterior region and lowest activity in the abdominal region. The activity in plucked (depilated) skin was only about 25% of that obtained from unplucked (depilated) tissue of the same region. D-Tryptophan, 5-hydroxytryptophan, and tryptamine were not degraded by the skin enzyme and the Km for L-tryptophan determined with the crude enzyme was 1 microM. The decycling activity of rat skin and liver for L-tryptophan began to be stimulated after birth and reached the highest level at 6 weeks. But, 1 week later, most of the skin activity suddenly disappeared and the low level continued at least until 12 weeks. In contrast, the hepatic enzyme did not change so drastically. These findings suggest that an enzyme that catalyzes L-tryptophan to L-kynurenine via L-formylkynurenine is present in rat skin.

Anthranilic acid metabolism in the isolated perfused rat liver: detection and determination of anthranilic acid and its related substances using high-performance liquid chromatography with electrochemical detection

In order to elucidate the anthranilic acid metabolism in animal tissue, the metabolism was studied in the isolated perfused liver of rats. A sensitive and rapid method was devised for determination of anthranilic acid and its related substances using high-performance liquid chromatography with electrochemical detection. 5-Hydroxyanthranilic acid and anthranilamide, which have not been detected in animal tissue, were found in the perfusate and in the bile secreted from the perfused liver, respectively. In addition, a non-enzymatic production of anthranilamide from anthraniloyl glucuronide in the presence of ammonium and bicarbonate ions was also observed. These present results suggest that, apart from undergoing glycine or glucuronide conjugation, anthranilic acid is metabolized to 5-hydroxyanthranilic acid and anthranilamide in the rat liver.

Isolation and properties of protein component as activator for anthranilamide hydroxylation in rat liver

Abstract A protein component, which activates the hydroxylation of anthranilamide by rat liver microsomal fractions in the presence of NADPH and air, was isolated from rat liver by DEAE-Sephadex and Sephadex G-75 column chromatography after heat denaturation and (NH4)2SO4 fractionation of the soluble fraction. The component contained one atom of copper and one atom of zinc per molecule. The molecular weight was 25 700. One tyrosine, six phenylalanine and no tryptophan residues were found per molecule. The activation of anthranilamide hydroxylation by the protein component was removed, when metals were separated from the protein. The apoprotein regained the activity after incubation with 5 μM CuSO4 in 50 mM Tris-HC1 buffer at pH 8.2. 5-Hydroxyanthranilamide and 3-hydroxyanthranilamide were formed in the enzymatic reaction in about equal quantities.

Rat Skin Tryptophan 2,3-Dioxygenase

The fact that a considerable amount of kynurenine (Kyn) is species-specifically accumulated in rat fur was discovered in 1960 by Ishiguro and co-workers (Hotta et al., 1960). The cause of this phenomenon has been exhaustively investigated in our laboratory, but no adequate explanation has been obtained so far.

INVOLVEMENT OF TRYPTOPHAN METABOLISM IN THE BODY COLOR OF CRUSTACEA

The terrestrial isopod, Armadillidium vulgare is usually grey or black in color, however, red ones are occasionally found in the field. This is caused by the mutation of the ommochrome genesis in the integument. We focused our experiments on the mechanism of pigment genesis in which tryptophan metabolism had been expected to be different from the grey or black wild types. We obtained the result that 3-hydroxyanthranilic acid content was significantly higher in the red phenotype than in the wild type, and kynureninase activity was also higher in the red phenotype.

Ommochrome Genesis in an Albino Strain of a Terrestrial Isopod

The contents of tryptophan (Trp) metabolites and the activities of the enzymes involved in ommochrome biosynthesis were measured in an albino strain of a terrestrial isopod Armadillidium vulgare. There was little difference between the Trp content in the albino mutant and that in the wild type, although the contents of 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA) and xanthommatin in the albino were significantly lower than those in the wild type. Tryptophan 2,3-dioxygenase (TDO) activity in the albino was extremely low, while the activities of Kyn-3-hydroxylase and kynureninase did not differ significantly between the two phenotypes. The extremely low activity of TDO is probably one of main reasons why almost no ommochrome pigment is produced in the albino mutant.