Chromatogram of Humoral Lectin of Bombyx mori by FPLC.

"Mono Q ion exchange chromatography was performed on Con A-adsorbed fractions from haemolymph and fat body of Bombyx mori, by means of FPLC system. The result showed that both of the chromatograms revealed similar patterns. On the other hand, Superdex 200 pg gel filtration was tried on the each Con A-adsorbed fraction and Mono Q ion exchange chromatography was performed on Fr-III obtained by means of gel filtration. The result showed that the elution pattern was much almost same as one of Mono Q ion exchange chromatography on the Con A-adsorbed fraction from the haemolymph or the fat body. Then, the result of SDS-PAGE analysis on the fractions showed that each lane gave a single band. These results strongly suggest that the humoral lectin-protein is produced and activated in fat body, and that it is moved into haemolymph. This research was partly supported by a grant from Tezukayama Gakuen.

Effect of Triton X 100 on Isolation of a Bombyx Humoral Lectin Activating Enzyme

Both the supernatant fraction and the precipitation one from the fat body of Bombyx mori showed the highest optical density when the density of Triton X 100 was one percent in 0.1M Tris-HCl buffer containing 0.1M NaCl. Accordingly, it was studied on gel filtration eluted with the buffer containing 1% Triton X 100 on a column of Superdex 200. The result showed that this method was more effective than the former method for obtaining the enzyme fraction from the fat body by gel filtration. Moreover, more enzyme fraction was obtained from larval fat body than from larval haemolymph on day 7 in the fifth instar by gel filtration. The fact showed the possibility that the change of neuraminidase activity due to the difference of time differed between the larval haemolymph and the fat body. This research was partly supported by a grant from Tezukayama Gakuen

Contribution of Glycosidases to Lectin Activity in Haemolymph of Bombyx mori

"The contribution of glycosidases, neuraminidase and galactosidase, to the lectin activity in the haemolymph of Bombyx mori was investigated. The neuraminidase activity was estimated according to determine sialic acid separated from N-acetyl neuramin lactose treated with the haemolymph of Bombyx mori, by means of high performance liquid chromatography (HPLC). Namely, the analysis of sialic acid was performed by using the ISA-07/S2504 column this time, while the analysis of sialic acid was performed by using the SCR 101H column until now. On the other hand, the galactosidase activity was estimated according to determine the content of D-galactose in the active lectin and in the inactive one by HPLC, comparatively. The results suggested that neuraminidase and galactosidase were activated with the ecdyson in the mature larval haemolymph of Bombyx mori, therefore that the appearance and the disappearance of the lectin activity were controled with these enzymes. This research was partly supported by a grant from Tezukayama Gakuen.

Tissue Distribution of Sialic Acid Relating to Humoral Lectin Activity of Bombyx mori

"The amout of sialic acid was determined in extracts of some tissues of Bombyx mori including haemolymph, haemocyte, fat body and midgut. The sialic acid was detectable in all the tissues by thiobarbituric acid assay. The amount of free sialic acid was large in larval haemolymph on day 3 and small in larval fat body on day 3 by HPLC. The haemagglutination activity of the Con A-adsorbed fraction was at comparable levels in fat body, midgut and haemolymph of mature larvae by the haemagglutination assay. Then, haemocyte and midgut were not detectable in pupal body. Moreover, the haemagglutination activity of the Con A-adsorbed fraction obtaied from fat body was at comparable levels in mature larvae and pupa by haemagglutination assay. These results strongly suggest that the humoral lectin-protein is produced and activated in fat body of Bombyx mori.

Research of a Bombyx Humoral Lectin Activating Factor by FPLC

"The result of the haemagglutination assay showed that the mature larval haemolymph fraction and the pupal fat body fraction had the ability to activate the inactive lectin-protein (130K-glycoprotein). The fact suggests the possibility that an enzyme activating the inactive lectin-protein presents in the fat body, that the inactive one is activated with the enzyme in the fat body, and that the active one is moved into the haemolymph. Accordingly, we tried gel filtration on a Superdex 200 column for researching neuraminidase in the fat body of Bombyx mori. The result of gel filtration showed that the crude enzyme fraction was obtained from the pupal fat body. Moreover, the result of the colour reaction by means of the thiobarbituric acid assay suggested the possibility that neuraminidase as the enzyme activating the inactive lectin-protein presented in the crude enzyme fraction obtained from the pupal fat body fraction by gel filtration. This research was partly supported by a grant from Tezukayama Gakuen.

A Study on Galactosidase Relating to a Bombyx Humoral Lectin Activity

"We studied on the method of the estimation of galactosidase activity in detail. Both of α-galactosidase activity and β-galactosidase activity were recognized in haemolymph and fat body during the fifth instar to pupae stage of Bombyx mori. It showed that the β-galactosidase activity was higer than α-galactosidase activity in all days. The activity was the highest on spinning stage. Moreover, we researched on galactosidase activity in fractions obtained from haemolymph by means of gel filtration. These results suggested the possibility that galactosidase might be present actually in the living body of Bombyx mori, and that galactosidase related to the Bombyx humoral lectin activity. This research was partly supported by a grant from Tezukayama Gakuen.

The Relationship between Sialidase Activity and Lectin Activity in Haemolymph of Bombyx mori

"A comparative study of the sialidase activity was performed by means of thiobarbituric acid assay and high performance liquid chromatography, using the JHA-treated larval haemolymph of Bombyx mori. The result showed that the sialidase activity increased at the larval maturation but decreased remarkably on spinning day. Accordingly, it was emphasized that the lectin activity in the haemolymph became highest after the terminal sialic acid in the sugar chains of the lectin-protein was removed with sialidase in vivo, because the content of binding sialic acid to the haemolymph protein decreased just after spinning and the lectin activity became highest just after spinning.

Characterization of a Bombyx Humoral Lectin Activating Enzyme

The result of gel filtration showed that the enzyme fraction was obtained from the pupal fat body. Both the result of the color reaction by means of the thiobarbituric acid assay and the result of HPLC analysis showed that the enzyme fraction affected N-ace-tylneuramin lactose which was a substrate against neuraminidase. These results suggested the possibility that neuraminidase exsisted in the enzyme fraction obtained from the pupal fat body by means of gel filtration, and that neuraminidase-like enzyme was isolated from the fat body by the methods described in this paper. This research was partly supported by a grant from Tezukayama Gakuen