Hiroya Kawasaki

Structural Proteins in the silkworm egg-shells

Abstract The major constituents of the egg-shell of the silkworm, Bombyx mori , were scleroproteins, and their disulphide linkages were the main force in stabilizing the organization of the shell. The scleroproteins contained three fractions, Proteins I, II, and III, in an approximate ratio of 76:23:1. Soluble derivatives of Proteins I and II were obtained in essentially homogeneous state. Protein II contained far larger amounts of cystine than did Protein I. Proteins I and II also differed in molecular size. Protein III differed from other fractions in composition and in solubility. Egg-shells of 5 silkworm species of the family Saturniidae were also investigated. All of them contained Protein I as the main component and Protein III in various amounts, but lacked Protein II.

Structural proteins in the egg envelopes of dragonflies, Sympetrum infuscatum and S. frequens

Abstract The egg envelopes of the dragonfly, Sympetrum infuscatum , consisted of three layers, the vitelline membrane (the innermost layer) and the inner and outer layers of chorion, their thickness being about 9, 0·3 and 3 μm. respectively. The major constituents of the envelopes were structural proteins, and they were divided into three different protein fractions. The two fractions which originated from the chorion were soluble in a thiol-urea solvent, and they were obtained as soluble S -carboxymethyl derivatives, CM-fractions I and II. Both of them contained a large amount of S -carboxymethylcysteine, but they differed from each other remarkably in the contents of amide, glycine, alanine and sugars. The average molecular weights of CM-fractions I and II were estimated to be 8900 and 15 000 respectively. The constituent of the vitelline membrane was insoluble in the thiol-urea solvent and was referred to as the ‘insoluble residue’. This protein fraction did not contain cystine at all but contained dityrosyl and trityrosyl residues (4 μmoles and 1 μmole per 100 mg. of sample respectively) as another type of intermolecular cross-link. The approximate proportions of CM-fractions I, II and insoluble residue in the egg envelopes in the native state were assumed to be 1 : 3 : 14. The yellow colour of the newly laid eggs changed to dark brown after exposure to air. This darkening process was found to occur in the vitelline membrane. The insoluble residue obtained from the dark eggs was also dark brown in colour and was more resistant against the hydrolysis by acid, alkali and proteolytic enzyme than was the yellow insoluble residue. The contents of tyrosine, lysine and histidine in the dark insoluble residue were markedly lower than those in the yellow residue, without any appreciable difference in the contents of other amino-acids. Similar changes were observable, though to a lesser extent, when the yellow insoluble residue was incubated with a mushroom tyrosinase. Thus, the chemical structure of the vitelline membrane, which had been stabilized by dityrosyl and trityrosyl residues, was assumed to be further reinforced during the darkening process by the mechanism of ‘self-tanning’. The results obtained from the eggs of another species of dragonfly, S. frequens , generally agreed well with those described for S. infuscatum material, with one exception that the CM-fraction II of S. frequens was obtained as two subfractions, CM-fractions IIa and IIb.

The identification of two N-acyldopamine glucosides in the left colleterial gland of the praying mantid, Tenodera aridifolia sinensis saussure, and their role in the oothecal sclerotization

Abstract Two glucosides were found in the left colleterial gland of Tenodera aridifolia sinensis Saussure, and they have been identified as 3- O - β -glucosides of N -acetyldopamine and N -malonyldopamine. β-Glucosidase activity was detected in the right colleterial gland. Sclerotized ootheca was extracted with 0.01 M acetic acid at about 20°C. The extract was found to contain a number of phenolic compounds modified particularly at the β-position of aglucones, which indicated that both of the aglucones acted as the sclerotizing agents, and suggested possible occurrence of the β-sclerotization in the mantid ootheca.

The identification of N-acyldopamine glucosides in the left colleterial gland of the praying mantids, Mantis religiosa L., Statilia maculata thunberg and Tenodera angustipennis saussure

Abstract Earlier work (Kawasaki and Yago, 1983, Insect Biochem. 13 , 267–271) has identified two glucosides, 3- O - β -glucosides of N -acetyldopamine and N -malonyldopamine, in the left colleterial gland of Tenodera aridifolia sinensis . In the present paper the glucosides of three other mantid species have been examined. The two glucosides of Tenodera angustipennis proved to be the same as those of T. aridifolia sinensis . The glucoside of Statilia maculata was 3- O - β -glucosyl- N -acetyldopamine, and that of Mantis religiosa 3- O - β -glucosyl- N -( N -acetyl- β -alanyl)dopamine. Thus, all of the mantid glucosides so far examined are 3- O - β -glucosides of N -acyldopamines.

Structural proteins in the egg envelopes of the mealworm beetle, Tenebrio molitor

Abstract The major constituents of the egg envelopes (the vitelline membrane and chorion) of the mealworm beetle, Tenebrio molitor , were structural proteins, and they were obtained as 3 different protein fractions. The fraction that was insoluble in a thiol-guanidine solvent was designated the insoluble residue, and the proteins dissolved in the solvent were converted to S -carboxymethyl derivatives and divided into 2 fractions of soluble proteins, CM-fractions I and II. CM-fraction I was essentially homogeneous as judged by the acrylamide gel electrophoresis, but CM-fraction II was heterogeneous. The solubility of the envelopes in the thiol-guanidine solvent indicated that the CM-fractions originated mainly from the chorion and the insoluble residue from the vitelline membrane. The proportion of the insoluble residue, CM-fractions I and II in the envelopes was assumed to be 2.5:1:2. Although CM-fractions I and II differed from each other in the solubility, sugar contents and molecular weight, they had a similar pattern of the amino-acid composition. Both of them contained a considerable amount of S -carboxymethylcysteine (3.6–4.6 moles per cent of the amino-acid residues) which had stabilized their structure as the disulphide bonds in the native state. On the other hand, the insoluble residue did not contain cystine at all but was stabilized by another intermolecular cross-link in the form of di- and trityrosyl residues (2.1 and 0.7 μmoles per 100 mg of sample, respectively), and the pattern of other amino-acids was clearly different from those of the soluble fractions. These results were discussed in comparison with other insect egg envelopes.

The identification of five N-acyldopamine glucosides in the left colleterial gland of the praying mantid, Hierodula patellifera serville

The glucosides so far identified in the left colleterial glands of four mantid species are 3-O-β-glucosides of N-acyldopamines, the acyl groups being acetyl, malonyl and N-acetyl-β-alanyl (Kawasaki and Yago, 1983, Insect Biochem. 13, 267-271; Yago et al., 1984, ibid. 14, 7–9). The gland of another mantid species, Hierodula patellifera, was found to contain as many as five glucosides, the three mentioned above and additional two that have been identified as 3-O-β-glucosides of N-β-alanyldopamine and N-(N-malonyl-β-alanyl)dopamine.

Conversion of serine to glycine during the formation of egg-shells in the silkworm, Bombyx mori

Abstract The egg-shell of the silkworm, Bombyx mori, contains water-insoluble protein(s) of which the glycine content is particularly high. The female adults before emergence thus require considerable amounts of glycine for the synthesis of the protein(s). As was the case in the spinning larvae of B. mori, serine was found to serve as a potent precursor of glycine in the female pharate adult and the conversion of serine to glycine proceeded mainly with the loss of the β-carbon of serine.

Structural proteins in the egg-shell of the horned beetle, Xylotrupes dichotomus

The egg-shell or chorion of the horned beetle consisted of three layers: the outer layer (about 6 μm thick) with irregular web-like structure, the middle layer (4 to 19 μm thick) made of many fine layers with some periodicity along and across the layers, and the homogeneous inner layer (1.2 μm thick). The inner layer and the inner part of the middle layer were continuous and other parts formed aeropyles. The major constituents of the chorion were structural proteins and the disulphide bonds of their cystinyl residues were the main force stabilizing the organization of the chorion. Most of the proteins were soluble in a thiol-urea solvent and were converted to soluble S-carboxymethyl derivatives (CM-fraction). The protein fraction, insoluble in the solvent, was designated the insoluble residue. Proteins in CM-fraction were fractionated into seven subfractions. The approximate proportion in the chorion of the eight protein fractions obtained, the insoluble residue, CM-fractions Ia, Ib, II, IIIa, IIIb, IIIc, and IIId, was assumed to be 2:18:16:1:3:3:2:1. The insoluble residue also contained a small amount of cystine (2.2 mole % of the total amino-acids as half-cystine), but its insolubility may be due to some aromatic tanning. CM-fractions differed from one another in the solubility, amino-acid composition and molecular weight. Their contents of S-carboxymethylcysteine varied markedly (4.5 to 21.5 mole %), and CM-fraction IIIs were unique in the high contents of both proline (13.9 to 27.7 mole %) and the cysteine derivative (8.9 to 21.5 mole %). CM-fraction Ia was found to originate from the outer layer. The crystalline middle layer consisted of four groups of proteins with quite different chemical nature, the native forms of CM-fractions Ib, II, and IIIa and the insoluble residue, whereas CM-fractions IIIb, IIIc, and IIId, low mol. wt proteins with the unique amino-acid composition, had organized the inner layer.

The tanning agent in the silk of the Japanese giant silkmoth, Dictyoploca japonica butler

The newly spun silk of Dictyoploca japonica contains a phenol oxidase and a tanning agent tentatively identified as N-(3,4-dihydroxyphenyllactyl)DOPA. Other saturniid silks so far studied contain an additional enzyme, β-glucosidase, and the tanning agents are present as glucosides of phenolic compounds (Brunet and Coles, 1974, Proc. R. Soc. Lond. B187, 133–170).

Enzymic activities involved in the oothecal sclerotization of the praying mantid, Tenodera aridifolia sinensis saussure

Abstract The enzyme(s) responsible for the sclerotization of mantid ootheca is secreted by the left colleterial gland. From an extract of the glands of Tenodera aridifolia sinensis , two soluble enzyme fractions of different activities were obtained. One fraction acted on N -acetyldopamine (NADA), a precursor of a representative sclerotizing agent, and produced NADA-quinone. The other did not act on NADA itself but converted the quinone to a highly reactive intermediate, such as quinone methide, which was able to react nonenzymically with nucleophilic compounds. Other insoluble enzyme preparations obtained from the silk and pupal cuticle of the Japanese giant silk moth, Dictyoploca japonica , also had these two activities.