"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.

Teruko Nakamura

Yasuo Kato

カトウ ヤスオ

ナカムラ テルコ

中村 照子

加藤 靖夫

P(論文)"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."departmental bulletin pape

加藤, 靖夫

カトウ, ヤスオ

Kato, Yasuo

中村, 照子

ナカムラ, テルコ

Nakamura, Teruko

Tezukayama University Repository

A Study on Galactosidase relating to a Bombyx Humoral Lectin Activity Yasuo KATo and Teruko NAKAMURA INTRODUCTION An animal lectin plays a significant role in a defense mechanism of living body such as cell adhesion or endocytosis (FUJITA, 2000; NOMURA et al., 2000). For instance, inver-tebrate lectins play a significant role in a non-specific self defense mechanism (KAWA-BATA, 2000) and in an apoptosis mechanism (HIRABAYASHI and KAsAl, 1998). Especially, humoral lectins of insects play a significant role through metamorphosis of insects and will be useful defense substances for human being in future (KOTANI et al., 1995; FUJITA et al., 1998; NATORI, 1998). On the other hand, AMANAI et al. (1994) suggested that a Bombyx lectin in haemolymph served some function in gonadal development. In a previous study, we reported that a humoral lectin-protein (130 K-glycoprotein) played a physiological role through metamorphosis of the silkworm, Bombyx mori, be-cause it always seemed to possess the highest lectin activity on spinning stage (KATO et al., 1994). We also reported on the appearance and the disappearance of the lectin activ-ity. We emphasized the possibility that the humoral lectin-protein was produced and ac-tivated in fat body of Bombyx mori, and that it was secreted into haemolymph (KATO et al . , 1998). Moreover, we researched a Bombyx humoral lectin activating factor in fat body by means of FPLC system, and obtained neuraminidase-like enzyme from the fat body (KATO and NAKAMURA, 1999, 2000). On the other hand, we have found that the ac-tive lectin disappeared the activity, when it was treated with galactosidase (KATO and NAKAMURA, 1987). This paper reports on galactosidase activity relating to a Bombyx humoral lectin ac-tivity, especially on a method of an estimation of galactosidase activity. This investiga-tion will provide useful information for understanding an original role of the lectin-pro-tein in vivo. -95-MATERIALS and METHODS 1. Preparation of samples A hybrid race, Shunrei X Shougetu, of the silkworm, Bombyx mori, was used in this experiment. Larvae were reared with fresh mulberry leaves. In preparing the samples for this research, haemolymph and fat body were collected. Haemolymph samples were col-lected by cutting the larval abdominal legs or pupal abdomen. Fat body samples were col-lected from dissected larvae or pupae, washed with cold 0.7% NaCl solution and homoge-nized in a glass homogenizer with a Teflon pestle. Mter centrifuging them at 3,000 rpm for 10 min at 4 'C, each of resultant supernatant was lyophilized. 2. Assay of galactosidase activity Galactosidase activity was assayed according to the method of LI and LI (1972), with slight modifications. p- nitrophenyl-a- galactopyranoside or p- nitrophenyl-p- galac-topyranoside was used as a substrate. One ml of 2 mM substrate in 0.05 M sodium cit-rate buffer (pH 4.0) was added in enzyme solution or sample solution. The reaction mix-ture was incubated at 37°C for 30 min. The reaction was stopped through 3 ml of 0.2 M borate buffer (pH 9.8). The mixture was measured for liberated p-nitrophenol by moni-toring the absorbance at 400 nm with a Shimadzu spectrophotometer type lN 1200. The scheme of the procedure was shown in Fig. 1. Procedure Larvae, Pupae Haemolymph Fat body Centrifuged Homogenized 3,000 rpm Centrifuged 10 min 3,000 rpm, 10 min Supernatant Supernatant ~ Dry freezed ~ Dry freezed a-galactosidase--2 mM p-nitrophenyl-a-galactopyranoside (in 0.05 M Sodium Citrate Buffer, pH 4.0) fi-galactosidase--2 mM p-nitrophenyl-{1-galactopyranoside (in 0.05 M Sodium Citrate Buffer, pH 4.0) ~ Incubation, at 37°C, for 30 min ~ 0.2 M Sodium Borate Buffer Solution (pH 9.8) Absorbance at 400 nm Fig. 1 Procedure of estimation -96-3. Gel filtration Gel filtration was performed using a Superdex 200 (Pharmacia) column (2.6 X 60 em) equilibrated with 0.1 M Tris-HCl buffer (pH 8.0) containing 0.1 M NaCl, at a constant flow rate of 150 mll hr. The effluent was collected in 5 ml fractions and measured at 280 nm with the spectrophotometer. RESULTS and DISCUSSION We studied on the method of the estimation of galactosidase activity in detail, ac-cording to the procedure shown in Fig. 1. Figure 2 shows a standard curve of ,a-nitro-phenol. The dilute solution of p-nitrophenol in 0.05 M sodium citrate buffer was colored through the addition of 0.2 M borate buffer, and then the absorbance was measured at 400 nm. It was confirmed that the optical density was directly proportional to the density of p-nitrophenol as shown in Fig. 2. Figure 3 shows the result of the study on a sub-strate of galactosidase. As shown in Fig. 3, it was confirmed that the color reaction looked no color in cases of only 0.2 M sodium borate buffer, ,o-nitrophenyl-11-galac-topyranoside in 0.05 M sodium citrate buffer with 0.2 M sodium borate buffer and 5 mg of dried matter of haemolymph in 0.05 M sodium citrate buffer with 0.2 M sodium borate buffer. On the other hand, it was confirmed that the color reaction looked yellow in cases 4 E c 0 0 ~ 3 +' «< CD C) c «< 2 .Q ... 0 fl) .Q ~ 20 100 Concentration (#I) Fig. 2 Standard curve of p-nitrophenol -97-200 4 E c 8 3 ~ ..... • G) 2 0 c • .a ... 0 1 fiJ .a c 0 CD ® @ @ @ Substrate Fig. 3 Activity of galactosidase against substrate CD : 0.2 M sodium borate buffer solution @: p-nitrophenyl-/1-galactopyranoside @ : 5 mg of dried matter of haemolymph @ @ : 5 mg of dried matter of haemolymph + p-nitrophenyl-/1-galactopyranoside ® : 10 mg of dried matter of haemolymph + p-nitrophenyl-/1-galactopyranoside ® : 20 mg of dried matter of haemolymph + p-nitrophenyl-p-galactopyranoside (j) : ft-galactosidase + p-nitrophenyl-/1-galactopyranoside (:tJ of 5 mg of dried matter of haemolymph in 0.05 M sodium citrate buffer containing p-ni-trophenyl-p-galactopyranoside with 0.2 M sodium borate buffer and /)-galactosidase in 0.05 M sodium citrate buffer containing p-nitrophenyl-;1-galactopyranoside with 0.2 M sodium borate buffer. The optical density increased as well as the increase of the density of the haemolymph, as shown in Fig. 3. Therefore, next experiment was performed using 5 mg of the dried matter of the haemolymph. It was also confirmed that each of p-nitro-phenyl-a-galactopyranoside and p-nitrophenyl-p-galactopyranoside was a substrate of a-galactosidase and /:?-galactosidase, respectively. Table 1 shows the result of the research of a-galactosidase activity and /1-galactosi-dase activity in haemolymph during the fifth instar to pupal stage of Bombyx mori. Both of the activities were recognized in haemolymph as shown in Table 1. It showed that p-galactosidase activity was higer than a-galactosidase activity in all days. The activity was the highest on spinning stage. Besides, it was guessed that the activity became high on the young day of the fifth ins tar, because galactosidase for the ecdysis of the fourth instrar remained high. Table 2 shows the result of the research of a-galactosidase activ-ity and i1-galactosidase activity in fat body during the fifth instar to pupal stage of Bom-byx mori. Both of the activities were recognized in fat body as shown in Table 2. Also, it showed that p-galactosidase activity was higer than a-galactosidase activity in all days. -98-Table 1 Activities of a-galactosidase and 13-galactosidase in haemolymph of Bombyx mori Activity of a-galactosidase Activity of ,3-galactosidase Absorbance Absorbance 3 rd day 0.226 3.010 6th day 0.148 1.637 8th day 0.157 1.724 9th day 0.189 1.755 lOth day 0.187 1.744 Pupae 0.263 1.614 Table 2 Activities of a-galactosidase and 13-galactosidase in fat body of Bombyx mon Activities of a-galactosidase Activities of 13-galactosidase Absorbance Absorbance 3 rd day 0.125 0.360 6th day 0.107 0.298 8th day 0.122 0.350 9th day 0.366 0.506 lOth day 0.117 0.298 Pupae 0.273 1.458 The activity was the highest on spinning stage, more distinctly than the occasiOn of haemolymph. Moreover, the. time of the highest activity in fat body seemed to be differ from one in haemolymph. Namely, the time of the highest activity in fat body seemed to be earlier than one in haemolymph. We reported previously on the relationship between galactosidase and haemagglutination activity of the Bombyx lectin in vitro ( 1987). Namely, the active lectin disappeared the activity, when it was treated with galactosi-dase. On that occasion, 11-galactosidase was more effective than a-galactosidase against disappearance of the lectin activity. The fact seemed to coincide with the result that 11-galactosidase activity was higer than a-galactosidase activity in all days as described above. Figure 4 shows an elution pattern of gel filtration on haemolymph of Bombyx mori on day 10 of the fifth instar. A column of Superdex 200 pg was equilibrated with 0.1 M Tris-HCl buffer containing 0.1 M NaCl (pH 8.0) and eluted with the same buffer. Three significant fractions, Fraction I, Fraction II and Fraction III were obtained by gel filtra-tion as shown in Fig. 4. Figure 5 shows the results of the research of a-galactosidase ac-tivity and 13-galactosidase activity in the three fractions obtained from haemolymph by gel filtration. As shown in Fig. 5, it was suggested that both of them were present in Fraction II. We reported previously that the active lectin-protein contained galactose as neutral sugar in its sugar-chain, and that galactosidase seemed to be present in haemo--99-2 I D m El 1.5 = 0 00 C"\1 +oJ «S Q) 1 u ~ ~ ~ 0 fll ~ 0. 5 < 10 20 30 40 50 60 70 Fraction number (5 ml/tube) Fig. 4 Elution pattern of gel filtration on a Superdex 200 column 0.4 ~ 0.3 0 0 ~ +oJ td Q) 0.2 C) a .0 J...l 0 fll .0 0. 1 < 0 Fraction I Fraction IT Fraction ill • Activity of {J -galactosidase D Activity of a -galactosidase Fig. 5 Activities of a-galactosidase and 11-galactosidase in the fractions obtained by gel filtra-tion on a Superdex 200 column lymph. However, they were reasoning on experimental results in vitro. Accordingly, it seems to be significance that the presence and activity of galacotosidase in haemolymph were recognized actually on experimental results in vivo as described above. In conclusion, we emphasize that galactosidase might be presents actually in the liv-ing body of Bombyx mori. The Bombyx humoral lectin was activated with neuraminidase -100-on spinning stage as described in the previous paper (KATO et al ., 1998). At that time, the lectin activity disappeared rapidly because galactosidase activity became high. We will try to elucidate reason why in future. SUMMARY We studied on the method of the estimation of galactosidase activity in detail. Both of a-galactosidase activity and /1-galactosidase activity were recognized in haemolymph and fat body during the fifth instar to pupae stage of Bombyx mori. It showed that the /1 -galactosidase activity was higer than a-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. REFERENCES A..\.IANAI, K., SuzuKI, Y. and 0HTANI, T. (1994): Involvement of a maternally transcribed lectin gene in the early development of Bombyx mori, Roux's Arch. Dev. Bioi., 203, 397-401. FuJITA, T. (2000): Innate defense lectin: Overview, PROTEIN, NUCLEIC ACID AND EN-ZYME, 45, 653-654. FuJITA, Y., KURATA, S., HoMMA, K. and NATORI, S. (1998): A novel lectin from Sarcophaga-its purification, characterization and eDNA cloning, J. Bioi. Chern., 273, 9667-9672. HIRABAYASHI, J. and KAsAl, K. (1998): Galectins and apoptosis, PROTEIN, NUCLEIC ACID AND ENZYME, 43,2442-2449. KATo, Y. and NAKAl\1URA, T. (1987) : Effect of glycosidase on lectin activity of glycoprotein in silkworm's haemolymph, J. of Tezukayama College, 24, 33-39. KATO, Y. and NAKAMURA, T. (1999): Characterization of a Bombyx humoral lectin activating en-zyme, J. ofTezukayama College, 36, 152-159. KATO, Y. and NAKAMURA, T. (2000): Effect of Triton X 100 on isolation of a Bombyx humoral lectin activating enzyme, J. ofTezukayama College, 37, 148-157. KATO, Y., NAKAMURA, T. and TAKEUCHI, T. (1994): Haemagglutination activity of haemolymph of Bombyx mori treated with a juvenile hormone analogue, J. Seric. Sci. Jpn., 63, 221-228. KATO, Y., NAKAMURA, T. and TAKEUCHI, T. (1998): Production and activation of humoral lectin protein in Bombyx mori, J. Seric. Sci. Jpn., 67, 319-326. KAwABATA, S. (2000): Nonself-recognizing lectins in invertebrate innate immunity, PROTEIN, NUCLEIC ACID AND ENZYME, 45, 679-689. KOTANI, E., YAMAKAWA M., IWAMOTO, S., TASHIRO M., MORI, H., SUMIDA, M., ]\IIATSUBARA, F., TA-.NIAI K., KADoNo-OKUDA, K., KATo, Y. and MoRI, H. (1995): Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor, Biochim. Biophys. Acta, 1260, 245-258.e. -101-LI, Y. -T. and LI, S. -C (1972): a -mannosidase, /3 -N-acetylhexosaminidase and /3 -galactosi-dase from Jack Bean Meal, "Methods in enzymology", ed. by V. Ginsburg, Academic Press, New York, 28, 702-713. NATORI, S. (1998): Insect lectins and defense mechanism, PROTEIN, NUCLEIC ACID AND ENZl'ME, 43, 2435-2441. NOMURA, K., MIZUGUCHI, 8., TAJIMA, T. and NOMURA, K. H. (2000): Blood group antigens and cell adhesion, SEIKAGAKU, 72, 91-104. -102-

A Study on Galactosidase Relating to a Bombyx Humoral Lectin Activity

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A Study on Galactosidase Relating to a Bombyx Humoral Lectin Activity

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