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Purification and distribution of a lectin in sea urchin (Anthocidaris crassispina) egg before and after fertilization
Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved 00 14.48?7/8 1/0900 I s-0?$0?.00/0
E.rperimentrrl Cell Resetrrch 135 (1981) 15-19
PURIFICATION
AND
DISTRIBUTION
(ANTHOCZDARIS
OF A LECTIN
CRASSISPZNA)
AFTER
IN SEA URCHIN
EGG BEFORE
AND
FERTILIZATION
HAJIME SASAKII and KENJI AKETA
SUMMARY A o-galactose specific lectin was purified by affinity chromatography from eggs of the sea urchin, Anrhocidaris crassispina. It is a dimer, composed of two monomers of 11500 MW. The highest affinity is directed to thiodigalactoside. Indirect immunofluorescent staining revealed the lectin as fluorescent spots, randomly distributed in the cytoplasm. After fertilization, these fluorescent foci become localized in the peripheral layer of the egg.
Lectins are known to be proteins which agglutinate erythrocytes by binding to specific saccharides on the cell surface [ 1, 21. They were found originally in various plant seeds, and then also in animal tissues [l61. Although their roles are not well understood, there are some studies which indicate regulatory roles in cells. For instance, the lectin in hepatic cell plasma membrane binds the glycoproteins to be digested in the parenchymal cells [4], and a lectin localized in the cortical granules of Xenopus lcrevis eggs is extruded from the granules after fertilization. It binds to the jelly layer surrounding the egg and renders the layer impenetrable to excess sperm [5]. We found a lectin in sea urchin eggs and purified it. In this report we describe the purification procedure and some properties of this lectin, with some information on its localization in the egg cytoplasm. MATERIALS
AND METHODS
Eggs of Anthcidaris cru.ssi.spintr were spawned into filtered sea water by injecting 5/9 M KC1 into its ?-RI1817
opened body cavity. They were washed several times with filtered sea water and were deiellied bv adiusting the pH of the suspending sea water to-5.0 “with 0.1 N HCI. After being washed several times with filtered sea water, they were dehydrated with several changes of acetone until the supernatant became colorless and transparent. The destained eggs were dried on a sintered glass funnel under reduced pressure and stored at -20°C until used. Acetone oowder of the eaas was susoended in IO vol of TBBS (Tris-buffere;- EDTA saline, 2 mM EDTA. 150 mM NaCl. 10 mM Tris-HCI. OH 7.4) and sonicated four times at 90 W for 15 set at intervals of 1 min with Ultrasonic Disruptor Model UR2OOP(Tomy Seiko, Tokyo). The supematant from centrifugation at I7 000 g for 20 min was collected and applied to an affinitv column composed of epoxvaciivated Sepharose 6B (Pharmacia, Uppsala, Sweden) coupled with thiodigalactoside (TDG, Sigma, St Louis. Mo). After the column was washed- with 10 vol TBES, the adsorbed lectin was eluted with 0.1 M lactose in TBES. The fractions with significant absorption at 280 nm were collected and concentrated in a Sartorius collodion bag SM 13200.The sample was further purified through a Sephadex G-100 column (I .5x 40 cm) equilibrated-with TBES. The purified lectin was stored in 0.1 M lactose-TBES at -20°C. Hemagglutinating activity was scored on a microtiter V-plate (Cooke Engineering, Virginia). In each well, 25.~1 of serial 2-fold dilutions of l&tin in TBES, 25 yl of 1%’ bovine serum albumin in TBS (Trisbuffered saline, 150 mM NaCl, 10 mM Tris-HCI. pH ’ Current address: Hopkins Marine Station, Stanford University, Department of Biological Sciences. Pacific Grove. CA 93950, USA.
16
Sasaki and Aketa
Table 1. Purification of A. crassispina egg lectin Total act.
Step
Titer
Protein (mglml)
Vol (ml)
Extraction Affinity chromatography Sephadex G-100
I 024
12.2
5
84
5 120
2 2
146 000 113 800
4 096 2 048
2 048 I 024
0.014 0.009
Sp. act.”
ClTiterlmg of protein. Initial em? acetone aowder was 500 ma. The decrease in specific activity is thought to be mainly due to the accurac&f the 2-foid dilution method.-
7.4), 25 ~1 TBES and 25 ~1 of trypsin-treated and fixed rabbit erythrocytes (TTFRE, see below) in TBS were mixed and kept at room temperature (24°C). The results were read 2 h later. For the saccharide specificity study, 25 ~1 of TBES in the reaction mixture was replaced by serial 2-fold dilutions of saccharide-TBES. The lectin solution was preadjusted to titer 16, and the minimal concentration of the saccharide required to inhibit the lectin activity was taken as measure of its inhibitory action. TTFRE was prepared as follows. Rabbit erythrocytes were collected from ear vein with a hypodermic syringe, washed by centrifugation in IO vol of TBS at 100 g for 5 min and made up to a 10% suspension in 0.1 % trypsin (Sigma, Type-HI)-TBS. After incubation at 37°C for 1 h, the cells were washed four times in 20 vol of PBS (phosphate-buffered saline, 150 mM NaCl, IO mM Na-phosphate, pH 7.4) and made to 10% suspension in I % glutaraldehyde-PBS. After I h incubation at 37”C, fixed cells were washed 4 times in 20 vol of PBS and incubated in 0.1 M glycine-PBS for another I h. The cells were finally washed in 20 vol of TBS and stored at 4°C. Protein was assayed by the method of Bradford [7] with bovine serum albumin as standard. SDS-polyacrylamide gel electro horesis (PAGE) was performed after the method of k eber & Osbotn [S] with modification of gel concentration to 12.5%. All chemicals used were of the highest grade available. Saccharides were purchased from Sigma, Difco (Detroit) and Wako (Tokyo). For the antibody production, the stained bands after SDS-PAGE of the afftnity-chromatographed preparations were cut out and used as antigens. Bands containing 40-60 wg of protein were homogenized in TBS, emulsified with an equal volume of Fteund’s complete adjuvant (Difco) and were injected subcutaneously in the back of a rabbit. Injections were repeated four times at one week intervals. The animal was bled a week after the last injection. Precipitin lines were observed according to Ouchterlony’s method [9]. The indirect immunofluorescence method was used for the localization of the lectin in the eggs. The eggs, both unfertilized and fertilized, were fixed in 10% formalin-sea water for 2 h at 25°C. To remove the fertilization membrane, inseminated eggs were transferred into I M urea just after the beginning of the membrane elevation. The fixed eggs were washed in
PBS and incubated in 20-fold dilution of anti-lectin serum for 30 min at 25°C. washed three times in 100 vol of PBS, incubated in 32-fold dilution of FlTClabeled anti-rabbit IgG goat IgG (Miles, Elkhart, Indiana) for 30 min at 25°C in the dark, washed three times in 100 vol of PBS and observed under the fluorescence microscope. In every staining, antiserum against the lectin of 23 000 MW was used.
RESULTS Affinity chromatography yielded a 1740fold purification with 80% recovery (table l), but SDS-PAGE of this preparation revealed two bands, one major and one minor Table 2. Minimal saccharide concentration necessary to inhibit acti\>ity of A. crcrssispina egg lectin Min. inhibitory cont.
Saccharide Thiodigalactoside p-Nitrophenyl-cY-o-galactoside P-Nitrophenyl-P-D-galactoside Methyl-cu-o-galactoside Methyl-P-o-galactoside o-Cialactose Lactose D-Fucose L-Arabinose N-Acetyl-u-galactosamine t.-Fucose D-Ribose D-Glucose o-Mannose o-Xylose
CrnM) 0.20 0.20 0.40 3.13 3.13 12.50 12.50 12.50 25.00 >50.0@ >50.00 >50.00 >50.00 >50.00 >50.00
Lectin titer was previously adjusted to 16.
Sen urchin
egg lectin
17
Fig. 2. SDS-polyacrylamide gel electrophoresis of affkitv ChromatoaraDhed oreoaration. 10% ael was used. -The prepa&& was treated either witch 0.1 rC SDS (4 or with 0.1 % SDS and I % B-mercantoethanol (h). Arrows indicate the additional minor components. See text. Fig. 1. SDS-polyacrylamide gel electrophoresis of purified lectin. 12.5c/cgel was used. ((I) After affinity chromatography. treated with 0.1% SDS. (b) After Sephadex G-100 gel filtration, treated with 0.1% SDS. (c) Same preparation as (b), treated with 1% p-mercaptoethanol and 0.1 % SDS.
lactose-TBES. Two estimations gave the identical value of 23000. When electrophoresed in an SDS-PAGE system after treatment with 1% @-mercaptoethanol, it behaved as a single band with estimated MW of 11500~700, whereas it was 23 0002 (fig. I LI). Further purification through 900 without P-mercaptoethanol pretreatSephadex G- 100 gel filtration eliminated the ment. Estimation was based on three exminor band, recovering a considerable periments. These results indicate that the amount of the activity (table 1, fig. lb, c). lectin is a dimer composed of the monomers The lectin showed high affinity to D- of the identical MW of 11500. galactoside, particularly to TDG, p-nitroThere was some variation between phenyl-a- and -P-D-galactoside (table 2). batches as to molecular weight in SDSHigher affinity was observed to the other PAGE, although in all cases these differgalactosides (except to lactose) than to D- ences were eliminated upon inclusion of galactose. Alteration in anomeric configura- /3-mercaptoethanol. There were usually two tion of the effective saccharides seems not bands, 23000 and 48000 MW, correspondto give rise to any significant change in the ing to the major and the minor respectiveaffinity of the lectin. Acetylated derivates ly, but two additional bands appeared in of D-g&CtOSt? such as ~-acetyl-D-galactossome batches (fig. 2~). Their MW were amine did not show any inhibitory effect 11500 and 35 000, which respectively corat a rather high concentration (50 mM). respond to that of monomeric and trimeric Molecular weight of this lectin was esti- forms of the lectin. Since all the compomated first by gel filtration through a Sepha- nents in these preparations were reduced in dex G-100 column equilibrated with 0.1 M the presence of ,&mercaptoethanol to a
18
Sasaki and Aketa
Fig. 3. Immunodiffusion test on the identity of antigenicity of the components of MW 23 000 and 48 000. Cenrrr ~rll, crude extract of acetone powder. Antiserum against the component of (a) MW 23000; (b) 48ooo.
single band of the molecular weight corresponding to that of the subunit of the lectin (fig. 2b), they may be monomer, dimer, trimer and tetramer, respectively. These results suggest that the subunit of the lectin tends to co-associate through disulfide bonds, to multiple forms, with the dimeric form being the most abundant, followed by the tetrameric form. The co-identity of the molecules at least
Fig. 4. Indirect immunofluorescence staining of fixed whole eggs ((11unfertilized; (h) fertilized. x600. EXP Cell Kc.v 13.5 (1981)
of the components of 23 000 and 48000 MW, was suggested by the result that antisera against these components fused with each other to form a single precipitin line (fig. 3). When fixed eggs were stained by the indirect immunofluorescence method, some particulate components in the cytoplasm were stained. They distributed randomly in the cytoplasm of unfertilized eggs, whereas in fertilized eggs, they were arranged in a layer at the peripheral region (fig. 4[1, h) and these do not correspond to cortical granules. In both cases, the eggs were not stained by preimmune serum. DISCUSSION The substance reported in this paper can be classified among the animal lectins, since its nature corresponds to that of lectins [ 1, 21; it can agglutinate erythrocytes with some saccharides as specific inhibitors. The lectin reported here is similar to those found in higher animals by Teichberg et al. and others [3, 10, 111in its sac-
Sea urchin egg lrctin
charide specificity, multiple forms and MW; those lectins specifically bind to TDG and lactose, have multiple forms and their dimer distributes in the range of 17000-33 000 MW [3, 10. Ill. Difference in lactose specificity of the present lectin from that of higher animals is of interest from the phylogenetic viewpoint. Indirect immunofluorescence staining did not show the specific localization of lectin in cortical granules, though this does not eliminate the possibility that lectin may occur in cortical granules. On the other hand, the redistribution of lectin subsequent to fertilization suggests two possibilities as to its major localization. The first possibility is that lectin is contained in or associated with some structural components in the egg cytoplasm. One possible example of this is the vesicles observed by Runnstrom [ 121 and Tanaka [ 131, which concentrate or localize to the peripheral region or cortex of eggs, as does lectin after fertilization. A second possibility is that the lectin molecules are not in granules but aggregate in the egg cytoplasm and migrate to the peripheral region subsequent to fer-
Printed
in Sweden
19
tilization. Immuno-electronmicroscopic observation, currently being undertaken, will answer these questions. We thank Professor D. Epel for his critical reading of the manuscript.
REFERENCES I. Sharon, N & Lis, H, Science 177 (1972) 949. 2. Nicolson, G L. Int rev cytol 39 (1974) 90. 3. Teichberg, V I, Silman, 1, Beitsch, D D & Resheff,
G, Proc natl acad xi US 72 (1975) 1383. 4. Paulson. J C. Hill. R L. Tanabe. T & Ashwell. G. J
bioi them 252 (1977) 8624. 5. Wyrick, R E, Nishimura, T & Hedrick. J L, Proc
natl acad sci US 71 (1974) 2067. 6. Muller. W E. Kurlec. B. Zahn. R K. Muller. I. Vaith, P & Uhlenbruck. G, J biol them 254 (1979) 7479. 7. Bradford. M M. Anal biochem 72 (1976) 248. 8. Weber. K & &born. M. J biol them 244 (1969) 4406. 9. Williams, C A & Chase, M W, Methods in imIO. II. 12. 13.
munoloev and immunochemistrv. Academic Press, New York (1971). deWaard, A. Hickman. S & Kornfeld. S. J biol them 251 (1976) 7581. Nowak, T P, Kobiler, D, Lawel, L E & Barondes, S H, J biol them 252 (1977) 6026. Runnstrom. J, Wilhelm Roux’ arch Entwicklungsmech organ I62 ( 1969)254. Tanaka, Y, Dev growth and differ 21 (1979) 331.
Received November 12, 1980 Revised version received March 20, 1981 Accepted March 26, 1981
E.rperimentrrl Cell Resetrrch 135 (1981) 15-19
PURIFICATION
AND
DISTRIBUTION
(ANTHOCZDARIS
OF A LECTIN
CRASSISPZNA)
AFTER
IN SEA URCHIN
EGG BEFORE
AND
FERTILIZATION
HAJIME SASAKII and KENJI AKETA
SUMMARY A o-galactose specific lectin was purified by affinity chromatography from eggs of the sea urchin, Anrhocidaris crassispina. It is a dimer, composed of two monomers of 11500 MW. The highest affinity is directed to thiodigalactoside. Indirect immunofluorescent staining revealed the lectin as fluorescent spots, randomly distributed in the cytoplasm. After fertilization, these fluorescent foci become localized in the peripheral layer of the egg.
Lectins are known to be proteins which agglutinate erythrocytes by binding to specific saccharides on the cell surface [ 1, 21. They were found originally in various plant seeds, and then also in animal tissues [l61. Although their roles are not well understood, there are some studies which indicate regulatory roles in cells. For instance, the lectin in hepatic cell plasma membrane binds the glycoproteins to be digested in the parenchymal cells [4], and a lectin localized in the cortical granules of Xenopus lcrevis eggs is extruded from the granules after fertilization. It binds to the jelly layer surrounding the egg and renders the layer impenetrable to excess sperm [5]. We found a lectin in sea urchin eggs and purified it. In this report we describe the purification procedure and some properties of this lectin, with some information on its localization in the egg cytoplasm. MATERIALS
AND METHODS
Eggs of Anthcidaris cru.ssi.spintr were spawned into filtered sea water by injecting 5/9 M KC1 into its ?-RI1817
opened body cavity. They were washed several times with filtered sea water and were deiellied bv adiusting the pH of the suspending sea water to-5.0 “with 0.1 N HCI. After being washed several times with filtered sea water, they were dehydrated with several changes of acetone until the supernatant became colorless and transparent. The destained eggs were dried on a sintered glass funnel under reduced pressure and stored at -20°C until used. Acetone oowder of the eaas was susoended in IO vol of TBBS (Tris-buffere;- EDTA saline, 2 mM EDTA. 150 mM NaCl. 10 mM Tris-HCI. OH 7.4) and sonicated four times at 90 W for 15 set at intervals of 1 min with Ultrasonic Disruptor Model UR2OOP(Tomy Seiko, Tokyo). The supematant from centrifugation at I7 000 g for 20 min was collected and applied to an affinitv column composed of epoxvaciivated Sepharose 6B (Pharmacia, Uppsala, Sweden) coupled with thiodigalactoside (TDG, Sigma, St Louis. Mo). After the column was washed- with 10 vol TBES, the adsorbed lectin was eluted with 0.1 M lactose in TBES. The fractions with significant absorption at 280 nm were collected and concentrated in a Sartorius collodion bag SM 13200.The sample was further purified through a Sephadex G-100 column (I .5x 40 cm) equilibrated-with TBES. The purified lectin was stored in 0.1 M lactose-TBES at -20°C. Hemagglutinating activity was scored on a microtiter V-plate (Cooke Engineering, Virginia). In each well, 25.~1 of serial 2-fold dilutions of l&tin in TBES, 25 yl of 1%’ bovine serum albumin in TBS (Trisbuffered saline, 150 mM NaCl, 10 mM Tris-HCI. pH ’ Current address: Hopkins Marine Station, Stanford University, Department of Biological Sciences. Pacific Grove. CA 93950, USA.
16
Sasaki and Aketa
Table 1. Purification of A. crassispina egg lectin Total act.
Step
Titer
Protein (mglml)
Vol (ml)
Extraction Affinity chromatography Sephadex G-100
I 024
12.2
5
84
5 120
2 2
146 000 113 800
4 096 2 048
2 048 I 024
0.014 0.009
Sp. act.”
ClTiterlmg of protein. Initial em? acetone aowder was 500 ma. The decrease in specific activity is thought to be mainly due to the accurac&f the 2-foid dilution method.-
7.4), 25 ~1 TBES and 25 ~1 of trypsin-treated and fixed rabbit erythrocytes (TTFRE, see below) in TBS were mixed and kept at room temperature (24°C). The results were read 2 h later. For the saccharide specificity study, 25 ~1 of TBES in the reaction mixture was replaced by serial 2-fold dilutions of saccharide-TBES. The lectin solution was preadjusted to titer 16, and the minimal concentration of the saccharide required to inhibit the lectin activity was taken as measure of its inhibitory action. TTFRE was prepared as follows. Rabbit erythrocytes were collected from ear vein with a hypodermic syringe, washed by centrifugation in IO vol of TBS at 100 g for 5 min and made up to a 10% suspension in 0.1 % trypsin (Sigma, Type-HI)-TBS. After incubation at 37°C for 1 h, the cells were washed four times in 20 vol of PBS (phosphate-buffered saline, 150 mM NaCl, IO mM Na-phosphate, pH 7.4) and made to 10% suspension in I % glutaraldehyde-PBS. After I h incubation at 37”C, fixed cells were washed 4 times in 20 vol of PBS and incubated in 0.1 M glycine-PBS for another I h. The cells were finally washed in 20 vol of TBS and stored at 4°C. Protein was assayed by the method of Bradford [7] with bovine serum albumin as standard. SDS-polyacrylamide gel electro horesis (PAGE) was performed after the method of k eber & Osbotn [S] with modification of gel concentration to 12.5%. All chemicals used were of the highest grade available. Saccharides were purchased from Sigma, Difco (Detroit) and Wako (Tokyo). For the antibody production, the stained bands after SDS-PAGE of the afftnity-chromatographed preparations were cut out and used as antigens. Bands containing 40-60 wg of protein were homogenized in TBS, emulsified with an equal volume of Fteund’s complete adjuvant (Difco) and were injected subcutaneously in the back of a rabbit. Injections were repeated four times at one week intervals. The animal was bled a week after the last injection. Precipitin lines were observed according to Ouchterlony’s method [9]. The indirect immunofluorescence method was used for the localization of the lectin in the eggs. The eggs, both unfertilized and fertilized, were fixed in 10% formalin-sea water for 2 h at 25°C. To remove the fertilization membrane, inseminated eggs were transferred into I M urea just after the beginning of the membrane elevation. The fixed eggs were washed in
PBS and incubated in 20-fold dilution of anti-lectin serum for 30 min at 25°C. washed three times in 100 vol of PBS, incubated in 32-fold dilution of FlTClabeled anti-rabbit IgG goat IgG (Miles, Elkhart, Indiana) for 30 min at 25°C in the dark, washed three times in 100 vol of PBS and observed under the fluorescence microscope. In every staining, antiserum against the lectin of 23 000 MW was used.
RESULTS Affinity chromatography yielded a 1740fold purification with 80% recovery (table l), but SDS-PAGE of this preparation revealed two bands, one major and one minor Table 2. Minimal saccharide concentration necessary to inhibit acti\>ity of A. crcrssispina egg lectin Min. inhibitory cont.
Saccharide Thiodigalactoside p-Nitrophenyl-cY-o-galactoside P-Nitrophenyl-P-D-galactoside Methyl-cu-o-galactoside Methyl-P-o-galactoside o-Cialactose Lactose D-Fucose L-Arabinose N-Acetyl-u-galactosamine t.-Fucose D-Ribose D-Glucose o-Mannose o-Xylose
CrnM) 0.20 0.20 0.40 3.13 3.13 12.50 12.50 12.50 25.00 >50.0@ >50.00 >50.00 >50.00 >50.00 >50.00
Lectin titer was previously adjusted to 16.
Sen urchin
egg lectin
17
Fig. 2. SDS-polyacrylamide gel electrophoresis of affkitv ChromatoaraDhed oreoaration. 10% ael was used. -The prepa&& was treated either witch 0.1 rC SDS (4 or with 0.1 % SDS and I % B-mercantoethanol (h). Arrows indicate the additional minor components. See text. Fig. 1. SDS-polyacrylamide gel electrophoresis of purified lectin. 12.5c/cgel was used. ((I) After affinity chromatography. treated with 0.1% SDS. (b) After Sephadex G-100 gel filtration, treated with 0.1% SDS. (c) Same preparation as (b), treated with 1% p-mercaptoethanol and 0.1 % SDS.
lactose-TBES. Two estimations gave the identical value of 23000. When electrophoresed in an SDS-PAGE system after treatment with 1% @-mercaptoethanol, it behaved as a single band with estimated MW of 11500~700, whereas it was 23 0002 (fig. I LI). Further purification through 900 without P-mercaptoethanol pretreatSephadex G- 100 gel filtration eliminated the ment. Estimation was based on three exminor band, recovering a considerable periments. These results indicate that the amount of the activity (table 1, fig. lb, c). lectin is a dimer composed of the monomers The lectin showed high affinity to D- of the identical MW of 11500. galactoside, particularly to TDG, p-nitroThere was some variation between phenyl-a- and -P-D-galactoside (table 2). batches as to molecular weight in SDSHigher affinity was observed to the other PAGE, although in all cases these differgalactosides (except to lactose) than to D- ences were eliminated upon inclusion of galactose. Alteration in anomeric configura- /3-mercaptoethanol. There were usually two tion of the effective saccharides seems not bands, 23000 and 48000 MW, correspondto give rise to any significant change in the ing to the major and the minor respectiveaffinity of the lectin. Acetylated derivates ly, but two additional bands appeared in of D-g&CtOSt? such as ~-acetyl-D-galactossome batches (fig. 2~). Their MW were amine did not show any inhibitory effect 11500 and 35 000, which respectively corat a rather high concentration (50 mM). respond to that of monomeric and trimeric Molecular weight of this lectin was esti- forms of the lectin. Since all the compomated first by gel filtration through a Sepha- nents in these preparations were reduced in dex G-100 column equilibrated with 0.1 M the presence of ,&mercaptoethanol to a
18
Sasaki and Aketa
Fig. 3. Immunodiffusion test on the identity of antigenicity of the components of MW 23 000 and 48 000. Cenrrr ~rll, crude extract of acetone powder. Antiserum against the component of (a) MW 23000; (b) 48ooo.
single band of the molecular weight corresponding to that of the subunit of the lectin (fig. 2b), they may be monomer, dimer, trimer and tetramer, respectively. These results suggest that the subunit of the lectin tends to co-associate through disulfide bonds, to multiple forms, with the dimeric form being the most abundant, followed by the tetrameric form. The co-identity of the molecules at least
Fig. 4. Indirect immunofluorescence staining of fixed whole eggs ((11unfertilized; (h) fertilized. x600. EXP Cell Kc.v 13.5 (1981)
of the components of 23 000 and 48000 MW, was suggested by the result that antisera against these components fused with each other to form a single precipitin line (fig. 3). When fixed eggs were stained by the indirect immunofluorescence method, some particulate components in the cytoplasm were stained. They distributed randomly in the cytoplasm of unfertilized eggs, whereas in fertilized eggs, they were arranged in a layer at the peripheral region (fig. 4[1, h) and these do not correspond to cortical granules. In both cases, the eggs were not stained by preimmune serum. DISCUSSION The substance reported in this paper can be classified among the animal lectins, since its nature corresponds to that of lectins [ 1, 21; it can agglutinate erythrocytes with some saccharides as specific inhibitors. The lectin reported here is similar to those found in higher animals by Teichberg et al. and others [3, 10, 111in its sac-
Sea urchin egg lrctin
charide specificity, multiple forms and MW; those lectins specifically bind to TDG and lactose, have multiple forms and their dimer distributes in the range of 17000-33 000 MW [3, 10. Ill. Difference in lactose specificity of the present lectin from that of higher animals is of interest from the phylogenetic viewpoint. Indirect immunofluorescence staining did not show the specific localization of lectin in cortical granules, though this does not eliminate the possibility that lectin may occur in cortical granules. On the other hand, the redistribution of lectin subsequent to fertilization suggests two possibilities as to its major localization. The first possibility is that lectin is contained in or associated with some structural components in the egg cytoplasm. One possible example of this is the vesicles observed by Runnstrom [ 121 and Tanaka [ 131, which concentrate or localize to the peripheral region or cortex of eggs, as does lectin after fertilization. A second possibility is that the lectin molecules are not in granules but aggregate in the egg cytoplasm and migrate to the peripheral region subsequent to fer-
Printed
in Sweden
19
tilization. Immuno-electronmicroscopic observation, currently being undertaken, will answer these questions. We thank Professor D. Epel for his critical reading of the manuscript.
REFERENCES I. Sharon, N & Lis, H, Science 177 (1972) 949. 2. Nicolson, G L. Int rev cytol 39 (1974) 90. 3. Teichberg, V I, Silman, 1, Beitsch, D D & Resheff,
G, Proc natl acad xi US 72 (1975) 1383. 4. Paulson. J C. Hill. R L. Tanabe. T & Ashwell. G. J
bioi them 252 (1977) 8624. 5. Wyrick, R E, Nishimura, T & Hedrick. J L, Proc
natl acad sci US 71 (1974) 2067. 6. Muller. W E. Kurlec. B. Zahn. R K. Muller. I. Vaith, P & Uhlenbruck. G, J biol them 254 (1979) 7479. 7. Bradford. M M. Anal biochem 72 (1976) 248. 8. Weber. K & &born. M. J biol them 244 (1969) 4406. 9. Williams, C A & Chase, M W, Methods in imIO. II. 12. 13.
munoloev and immunochemistrv. Academic Press, New York (1971). deWaard, A. Hickman. S & Kornfeld. S. J biol them 251 (1976) 7581. Nowak, T P, Kobiler, D, Lawel, L E & Barondes, S H, J biol them 252 (1977) 6026. Runnstrom. J, Wilhelm Roux’ arch Entwicklungsmech organ I62 ( 1969)254. Tanaka, Y, Dev growth and differ 21 (1979) 331.
Received November 12, 1980 Revised version received March 20, 1981 Accepted March 26, 1981