Yoshishige Umebachi

Beta-alanine as a constituent of the dopamine derivative, SN-1 of Papilio xuthus

Abstract 1. 1. The β-alanine-containing DOPAmine derivative, SN-1, was purified from the extract of the wings of P. xuthus. 2. 2. The purified SN-1 was arylated with 1-fluoro-2,4-dinitrobenzene, and after hydrolysis, DNP-β-alanine was found. The infrared spectrum of SN-1 showed the presence of amide bond. The β-alanine proved to be released by a very mild hydrolysis. 3. 3. From the previous and present results, it has been presumed that the SN-1 is a N-(β-alanyl)DOPAmine derivative.

Further studies on the dopamine derivative, SN-1 derived from the yellow pigments of Papilio xuthus

For the purpose of purifying the dopamine derivative SN-1 which had been reported as a decomposition-product of the yellow pigments of Papilio xuthus, the isolation of the substance with column chromatography was tried. The crude extract from wings or yellow scales was heated in 10−3 N HCl and chromatographed on the Amberlite CG-50 column. On this column, kynurenine and an unknown o-diphenolic substance were clearly separated. The latter coincided with the SN-1 and was re-confirmed to be a dopamine derivative using 14C-dopamine. On the basis of these observations, the purification of SN-1 was performed through the DEAE-Cellulose, Amberlite CG-50, and Biogel P-2 columns. The purified SN-1 was hydrolyzed in 1 N HCl, and the hydrolysates were again chromatographed on the Biogel P-2 column, in which two main products were found. One has been identified as β-alanine. The other was a dopamine derivative closely related to arterenol. It has been presumed that SN-1 is a dopamine derivative containing β-alanine in the side chain. The contents of SN-1 and kynurenine in wings were estimated during the pharate adult stage. It was shown that both substances increase in parallel with each other during the yellow pigment formation. In addition to the above β-alanine, this amino acid was found also in the scales remaining after the yellow pigments and other water-soluble substances were extracted.

Clear evidence for the presence of β-alanine as a constituent of papiliochrome II

Abstract 1. 1. From the crude extract of the wings of Papilio xuthus labelled with [1-14C]β-alanine, the decomposition-product SN-1 of Papiliochrome II was purified and hydrolyzed. Of two main hydrolysates, one was identified as [14C]β-alanine, and the other was the non-radioactive substance SN-1a. 2. 2. The above results were confirmed by the decomposition of purified Papiliochrome II and by the autoradiograph of thin-layer chromatograms of Papiliochrome II and its decomposition-products. 3. 3. It was also proved using [1-14C]DOPAmine that SN-1a is a DOPAmine derivative. 4. 4. From the previous and present results, it is evident that Papiliochrome II contains, as constituents, both kynurenine and a DOPAmine derivative containing β-alanine.

Synthesis, accumulation and excretion of kynurenine during the pupal and adult stages of Papilio xuthus

Abstract Changes of kynurenine levels in fat body, haemolymph and wings during the pupal stage of Papilio xuthus were determined. In fat body and haemolymph, free kynurenine begins to increase at the time of the onset of eye pigmentation, reaches a maximum at the stage of, or shortly after red spot appearance in the wings and then rapidly decreases. The bound form of kynurenine seems not to be present. In the wings, on the contrary, the bound form of kynurenine begins to increase shortly before the appearance of red spot and accumulates up to the time of emergence. Tryptophan oxygenase activity is greatest in the fat body, with less in the wings and haemolymph did not show any activity. At the emergence of the butterfly, a considerable amount of kynurenine is excreted in the meconium. Empty pupal cases also contain some kynurenine. At the same time in the adult, the yellow scales contain the largest amounts of kynurenine, and the adult body (minus yellow scales) also contain a considerable amount of kynurenine. All these results suggest the possibility that kynurenine is synthesized in the fat body, is transported through the haemolymph and accumulates as the bound form in the yellow scales. But the possibility cannot be ruled out that some kynurenine is synthesized in the wings.

Some properties of the kynurenine present in wing-scales of Papilio butterflies

Abstract 1. 1. 14 C-Tryptophan was incorporated as 14 C-kynurenine into the pale yellow, deep yellow and reddish brown scales of Papilio butterflies. 2. 2. The quantity, solubility and stability of kynurenine were compared among these three kinds of scales. 3. 3. The kynurenine present in reddish brown scales is smaller in quantity, more slightly soluble and more stable than that of pale and deep yellow scales. 4. 4. The pigment of the reddish brown scales is soluble in 1 N NaOH and precipitates by being acidified. But most of the kynurenine is lost during this procedure.

Accumulation and excretion of beta-alanine in Papilio xuthus (lepidoptera: Papilionidae)

Abstract 1. 1. The accumulation and excretion of free, soluble-bound, and insoluble-bound β-alanine during the pupal and adult stages of P. xuthus were investigated. 2. 2. Free β-alanine increases from the middle pupal stage, reaches a maximum at the time of red-spot appearance, and then decreases down to the time of emergence. 3. 3. The soluble-bound β-alanine sharply increases shortly before emergence and accumulates in the pale yellow scales of adult. 4. 4. The insoluble-bound β-alanine rapidly increases in the late pupal stage and accumulates in the wing-membrane and body wall of adult. 5. 5. The meconium contains a small amount of soluble-bound β-alanine, while the exuviae contain only a trace of β-alanine.

Further studies on dopamine and N-acyldopamine during the pupal stage of Papilio xuthus (Lepidoptera: Papilionidae)

Abstract 1. 1. Level changes of free and soluble-bound forms of dopamine (DA), N-β- alanyldopamine (NBAD), and N- acetyldopamine (NADA) were studied in the haemolymph and wings during the pupal stage of P. xuthus. 2. 2. In the haemolymph, free and soluble-bound forms of DA and NBAD are present only in small quantities throughout the pupal stage. As for NADA, the free form is absent, while the soluble-bound form showed a medium-sized peak at the middle pupal stage. 3. 3. In wings, the soluble-bound form of DA and NBAD was absent throughout the pupal stage. On the other hand, free DA showed a high and sharp peak shortly before emergence, and free NBAD showed a high and broad peak just at the time of pale yellow pigment formation. As to NADA, both free and soluble-bound forms were absent during the late pupal stage. 4. 4. The soluble-bound form of NADA in haemolymph has been identified as 4-O-β- glucoside . 5. 5. These results indicate that the precursor of papiliochrome II is the free form of NBAD.

Chemical properties of the orange pigment of Lycaena Phlaeas (Lepidoptera)

Abstract 1. 1. Some chemical properties of the orange pigment of L. phlaeas were investigated by thin-layer chromatography, hydrolysis and absorption spectra. 2. 2. On hydrolysis, the pigment released a white-blue fluorescent substance which was identified as 3-hydroxyanthranilic acid. All the experimental results indicate that the pigment is a 3-hydroxyanthranilic acid derivative. 3. 3. The pigment seems to be extracted from scales as a protein-bound molecule, because the hydrolysate contained ordinary protein-constituent amino acids. Interestingly, β-alanine was also found.

Kynurenine, β-alanine, and dopamine in the deep yellow pigment of Papilio machaon (lepidoptera: papilionidae)

Abstract 1. 1. The deep yellow pigment extracted with 1% HCl methanol has been proved to decompose to kynurenine and N-β- alanylnorepinephrine (NBANE) by being heated in dilute hydrochloric acid 2. 2. NBANE decomposes further to norepinephrine and β-alanine by hydrolysis in 1,2 N hydrochloric acid. The decomposition was a little more difficult than in Papiliochrome II. 3. 3. The molar ratio of β-alanine to kynurenine was about two. Among amino group of total β-alanine, only 42.5% was free, and the remaining is probably bound to some other group. 4. 4. From the HCl-methanol extract, three kinds of pigment fractions were obtained, and all of them showed the same decomposition-products.

Some chemical properties of the HCl-methanol extract from the puparial cuticle of Drosophila melanogaster

1. The HCl-methanol (HCl-MeOH) soluble fraction from the puparial cuticle of yellow, black and ebony of D. melanogaster was hydrolyzed in hydrochloric acid and examined for beta-alanine, ketocatechol, and acetic acid. 2. Between beta-alanine and ketocatechol and between beta-alanine and acetic acid, a quantitatively inverse relationship was found, respectively. The former relationship was further confirmed by the feeding experiment of beta-alanine to black. 3. Of total beta-alanine in the HCl-MeOH extract, the proportion of those having free amino group was 74.8 per cent. 4. All these results indicate that the HCl-MeOH soluble fraction of the puparial cuticle may be useful for investigating the cross-link structure of the cuticle.