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Lytic Activity and Biochemical Properties of Lysozyme in the Coelomic Fluid of the Sea UrchinStrongylocentrotus intermedius
Journal of Invertebrate Pathology 73, 214–222 (1999) Article ID jipa.1998.4808, available online at http://www.idealibrary.com on
Lytic Activity and Biochemical Properties of Lysozyme in the Coelomic Fluid of the Sea Urchin Strongylocentrotus intermedius Motohiro Shimizu,* Shin Kohno,* Hisashi Kagawa,* and Nobutoshi Ichise*,1 *Laboratory of Comparative Physiology, Department of Biology, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan Received July 11, 1997; accepted July 14, 1998
Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11° and 25°C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS–PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. r 1999 Academic Press Key Words: Strongylocentrotus intermedius; lysozyme; coelomic fluid; coelomocyte extract; immunoblotting; purification; Biogel filtration; affinity chromatography; biodefense mechanism.
INTRODUCTION
Lysozyme, known as a lytic enzyme attacking grampositive bacilli, is present in various tissues of animals and plants. This enzyme is contained in the kidney, spleen, digestive tract, and mucus of epidermis in animals (Audy et al., 1989); these organs are at risk in bacterial infection. On the other hand, stress and disease in animals affect the lytic activities of lyso-
1 Present address: Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido 060, Japan.
0022-2011/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
zymes (Jolle`s and Jolle`s, 1984; Mo¨ck and Peters, 1990). Lysozyme may be involved in the biological defense system through not only bactericidal activity but also other procedures (Studnicka et al., 1986; Mo¨ck and Peters, 1990). We have a little information on this lytic enzyme in echinoderms, although several kinds of substances participating in biodefense mechanisms, such as lectins, hemolysins, aggultinins, and others, have been identified (Wardlaw and Unkles, 1978; Parrinello et al., 1979; Canicatti, 1987, 1989, 1991, 1992; Canicatti and Ciulla, 1987; Azumi et al., 1991; Stabili et al., 1996; Roch, 1996). Jolle`s and Jolle`s (1975) studied the biochemical properties of lysozyme in the sea star Asterias rubens. In the sea cucumber Holothuria polii, lysozyme-like lytic activity was also found in coelomocytes (Canicatti et al., 1989). Canicatti and D’Ancona (1990) also confirmed lysozyme in the mucus of the sea star Marthasterias glacialis. In the sea urchin Paracentrotus lividus, Stabili and Canicatti (1994) identified this enzyme as one of the antibacterial factors in the seminal plasma. However, we have less information on this lytic enzyme in the coelomic fluid of sea urchins. Recently, ‘‘spotting disease’’ or ‘‘spine-shedding disease’’ has been found in sea urchins living on some coasts and in breeding stations in Japan (Hamaguti et al., 1993; Shimizu et al., 1995; Tajima et al., 1997). Similar urchin diseases have been reported in urchins living in several ocean coasts of Europe, Canada, and other areas (Jangoux, 1984; Maes and Jangoux, 1984; Scheibling and Stephenson, 1984). These diseases cause high motality rates. In the Akauni (red sea urchin) Pseudocentrotus depressus affected by the spineshedding disease, a bacillus, Frexbactor sp., was identified as a pathogen (Hamaguti et al., 1993). A similar bacillus was also found in spotting disease of Strongylocentrotus intermedius and is supposed to be a pathogen of the disease (Shimizu et al., 1995; Tajima et al., 1997). The latter species lives in northern Japan and falls to the disease in high temperature seasons, but the former type, living in southern Japan, does so in low temperature seasons. The bacillus infection and unpro-
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pitious temperatures for the urchins may affect their biodefense system. Therefore, if they have lysozyme, its lytic activity may respond to these environmental factors. In the present study, we identified lysozyme in the coelomic fluid, including coelomocytes, in S. intermedius, examined its lytic activity in urchins held at the two different water temperatures, and then clarified some biochemical properties of this enzyme. Furthermore, we tried to purify the urchin lysozyme and identify the immunological resemblance between this enzyme and hen egg white lysozyme by immunoblotting. MATERIALS AND METHODS
Experimental Animals About 150 specimens (50 g and 56 mm in mean body weight and test diameter, respectively) of S. intermedius were used in this study. They were collected from the tidal zone of the coast near Hakodate in spring and summer (environmental seawater temperatures, about 5–20°C) and reared in the Usujiri Experimental Marine Station of Hokkaido University near Hakodate until use. Ten individuals each were held at the two different water temperatures of 11° and 25°C, respectively, for 4 weeks in aquaria (30 ⫻ 20 ⫻ 30 cm) equipped with a circulating filter system. The seawater used was filtered with a mesh of 1 µm pore size (Organo PF Model III). Wakame, Undaria, fresh or desalted from salted ones, was given as food every 2 days. Preparation of the Samples for Lysozyme Assay Coelomic fluid was withdrawn using plastic spoids from the coelom by puncture of the peristomial membrane into a 10-ml plastic test tube and then centrifuged at 3000 rpm for 10 min to precipitate coelomocytes. The supernatants of three to five tubes were pooled and filtered with a paper filter (Toyo No. 6), dialyzed against 0.67 M phosphate buffer (pH 6.8) for 6 h, and lyophilized as the sample of acellular coelomic fluid. The precipitates of those tubes were pooled and homogenized with 3–5 ml phosphate buffer and centrifuged at 15,000 rpm for 10 min to separate the supernatant as the sample of coelomocyte extract. These samples were stored at ⫺20°C until use.
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Fifty microliters of the specimens was mixed with 3 ml of the medium. At first, the samples were incubated at 11°, 20°, and 37°C for 15, 90, and 240 min to determine the optimal incubation temperature and time of its lytic activity. Lytic activity was detected by measuring the absorbancy at 540 nm. Based on the results of these preliminary experiments, the following incubations were done at 37°C for 4 h. Optimal pH in lytic activity was determined at various pHs using medium containing 100 mM NaCl and optimal NaCl concentration done at pH 7.0 using media containing NaCl at various concentrations (0–200 mM). To examine the availability of hen egg white lysozyme (HEWL) as the standard substance in the measurement of the urchin lysozyme, HEWL lytic activity was preliminarily measured using media containing NaCl at various concentrations (0, 100, and 200 mM), for 15 min at pH 7.0 and 37°C. Electrophoretic Examination of Lysozyme Fraction Lysozyme fraction was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) by the method of Audy et al. (1989). The gel (15%) contained 0.4% Micrococcus lysodeikticus. Each sample (lyophilized coelomic fluid, coelomocyte extract, and HEWL) was mixed with the same volume of a sample buffer (0.01% Tris–HCl, pH 6.8) containing 0.1% SDS and 2% sucrose and boiled for 2 min. It was electrophoretically fractionated by SDS–PAGE at 30 mA in accordance with the method of Laemmli (1970). After electrophoresis, the gels were incubated with an activating buffer (50 mM sodium phosphate, 100 mM NaCl, 1% Triton X-100, pH 6.5) in a sealed well at 37°C for 4 h. Lytic zones with lysozymes were detected under a black background: the zone was observed to be transparent. Purification of Sea Urchin Lyzozyme About 120 individuals were used for purification of the lysozyme of the coleomic fluid and coelomocytes.
Measurement of Lysozyme Activity Lytic activity of lysozyme was measured spectrophotometrically by the method of Smolebis and Hartsell (1949). Twenty-four micrograms of dried M. lysodeikticus was mixed with 100 ml 0.67 M phosphate buffer, kept for 2 days, and then centrifuged. The supernatant was used as the medium for lysozyme measurement.
FIG. 1. Lysozyme fractions in SDS–PAGE of the acellular coelomic fluid and coelomocyte extract of S. intermedius. 1, hen egg white lysozyme (HEWL); 2–5, coelomocyte extract; 6, 7, acellular coelomic fluid.
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FIG. 2. Effects of incubation temperature and time on the lytic activity of lysozyme in coelomocyte extracts of S. intemedius.
The lyophilized acellular coelomic fluid and the coelomic extract were obtained by the method described before. Precipitation by saturating ammonium sulfate or ethanol. At first, the samples were salted out with ammonium sulfate at 35, 50, 75, and 90% saturation. The precipitated proteins in each saturated state were collected by centrifugation (10,000g, 20 min, 0°C) and dissolved to 0.05 mol/liter So¨rensen phosphate buffer (pH 6.8). As the secondary, the samples were brought to 20% saturation with ethanol, stirred for 1 h, and then centrifuged at 10,000g for 20 min. The supernatant was then adjusted to 80% saturation with additional etha-
FIG. 3. Effect of pH on the lytic activity of lysozyme in coelomocyte extracts of S. intermedius.
FIG. 4. Effect of NaCl concentrations on the lytic activity of lysozyme in coelmocyte extracts of S. intermedius.
nol and stirred for 1 h, and the precipitated proteins were collected by centrifugation as above. Gel filtration with Biogel P-30. The supernatant at 20% ethanol saturation was layered on a Biogel P-30 column (3 ⫻ 70 cm) equilibrated with 0.05 mol/liter So¨rensen phosphate buffer (pH 6.0), eluted with the same buffer at 0.4 ml/s, and collected at 4 ml in each tube. The combination method of gel filtration (Biogel P-30) and affinity chromatography (heparin Sepharose HP). The coelomocyte extracts obtained from 23 individuals were dialyzed against 0.05 mol/liter phosphate buffer (pH 6.0) and then applied to the Biogel P-30 column equilibrated with the same buffer. They were eluted with the same buffer at one drop/7 s, and 4 ml was collected in each tube. The fractions having lysozymal lytic activity were pooled and applied to the heparin
FIG. 5. Effect of NaCl concentrations on the lytic activity of hen egg whate lysozyme.
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adjuvant (Wako Pure Reagent Co. Japan) to form an emulsion. The emulsion was subcutaneusly injected into a rabbit six times once a week. When a sufficient formation of antibody was confirmed by using a little blood collected from an epiotic vein, whole blood was obtained by puncturing the heart. The blood was kept for 1 h at 37°C and overnight in a refrigerator (4°C) and then centrifuged at 3000 rpm for 20 min at 4°C to obtain the supernatant as anti-HEWL rabbit serum. The antiserum was kept ⫺80°C until use.
FIG. 6. Lytic activities of lysozyme in acellular coelomic fluid and coelomocyte extracts of S. intermedius acclimated to different water temperatures (11° and 25°C). The activity of acellular coelomic fluid was not determined at 25°C. The result is expressed as the mean ⫾ SE (bar).
Sepharose HP column equilibrated with the phosphate buffer. The protein fractions were eluted stepwise with the same buffer containing 0.1–1 mol/liter NaCl at a drop/4 s, and 2 ml each was collected. The peak fractions were pooled and applied to SDS–PAGE to determine the purification state of the lysozyme fraction. The lysozymal lytic activity of each fraction in all purification steps was measured by absorbancy at 540 nm and the protein content was done by the method of Bradford (1976) or absorbancy at 280 nm, and then a specific activity (OD 540 nm/µg protein), yield (volume %, protein), and purification (fold, lytic activy) were calculated in each fraction. Analysis of Immunological Similarity between Sea Urchin Lysozyme and HEWL Formation of anti-serum against HEWL. Five hundred micrograms of HEWL (Sigma) was dissolved in 1 ml of 0.05 mol/liter phosphate buffer (pH 6.0) and then mixed with the same volume of Freund’s complete
Immunoelectrophoresis. Immunoelectrophoresis of the coelomocyte extracts was performed by the method of Grabar and Williams (1953). The gel was made of Veronal acetate buffer (10 mM Veronal, 50 mM sodium Veronal; pH 8.6) containing 1% agarose. The extracts were electrophoresed at a constant current, 3.6 mA/cm, until a running front reached 3 cm long from the starting point. When the electrophoresis was finished, a groove was made on the gel parallel to the running sample. One hundred twenty microliters of the antiserum was applied to the groove and kept overnight. After being washed with a physiological solution and distilled water, the gel was dried and then stained with Coomassie brilliant blue (CBB) staining solution (0.1% CBB R-250 in a mixture of ethanol:acetic acid:distilled water 1:1:1). Immunoblotting. The coelomocyte extract and HEWL were respectively dissolved in 0.05 mol/liter phosphate buffer (pH 6.0) to 5 mg/ml final concentration and then applied to SDS–PAGE in accordance with the methods of Laemmli (1970). After electrophoresis, the fractionated protein bands were transferred to polyvinyldendifuruolid membrane (PVDF, Millipore) by the method of Towbin et al. (1979). They were blocked for 1 h with 5% skim milk with Tris-buffered sodium chloride solution (TBS: 20 mM Tris, 500 mM NaCl, pH 7.5) and then reacted with diluted primary antiserum (anti-HEWL rabbit serum) at 37°C for 1 h. After the PVDF was washed briefly with distilled water, twice 10 min each with 0.05% Tween 20 in TBS (TTBS), and 10 min with TBS, it was treated with the secondary antibody (horseradish peroxidase-labeled
TABLE 1 Protein Contents and Lytic Activities of Lysozyme Fractions Salted out with Ammonium Sulfate in Acellular Coelomic Fluid of S. intermedius Fraction Acellular coelomic fluid Ammonium sulfate saturation 0–35% 35–50% 50–75% 75–90%
Total protein (µg)
Total lytic activity (OD 540 nm ⫻ 103 )
Specific activity (OD 540 nm ⫻ 103/ µg Pr)
5900
5380
0.912
500 590 670 190
130 720 1650 340
0.259 1.221 2.466 1.813
Yield (V%, protein) 100 8.4 10.6 11.3 3.2
Purification (fold, lytic activity) 1 0.28 1.34 2.70 1.99
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TABLE 2 Lysozyme Fractions Isolated by Ethanol Saturation in Acellular Coelomic Fluid and Coelomocyte Extracts of S. intermedius Fraction
Total protein (µg)
Total lytic activity (OD 540 nm ⫻ 103 )
Specific activity (OD 540 nm ⫻ 103/ µg Pr)
Yield (V%, protein)
Purification (fold, lytic activity)
Acellular coelomic fluid (crude) 20–80% ethanol sediment Coelomocyte extract (crude) 20–80% ethanol sediment
135,000 2,870 71,880 2,920
278,400 2,200 80,940 8,530
2.1 7.6 1.13 2.92
100 2.6 100 4.1
1.0 3.6 1.0 2.6
anti-rabbit IgG goat serum, Sigma A-9169) for 2 h at room temperature. Consecutively, the membrane was washed twice with TTBS (10 min each) and TBS (10 min) and colored with a mixture of 0.06% 4-chloro-1naphtol, 0.01% hydrogen oxide, and 20% methanol. The membrane was dried after wash with distilled water. RESULTS
Lysozyme Fractions of Acellular Coelomic Fluid and Coelomocytes of S. intermedius In SDS–PAGE, a clear lytic zone was found in the coelomocyte extract (Fig. 1). The colomic fluid showed a faint zone at the same position. Both lysozyme zones were situated at about 14 kDa, being a little smaller than HEWL (14.5 kDa) in molecular weight. Identification of an Optimum Method for Measuring the Lytic Activity of Urchin Lysozyme Lysozyme of the coelomocyte extract grew in its lytic activity as the increase in the examined incubation temperature and time; the highest activity was obtained at 37°C for 4 h (Fig. 2). The lytic activities were high at pH 6.5–7.0 and decreased in ranges lower than pH 5 and higher than pH 8 (Fig. 3). Its activity was maximum when the incubation medium contained 100 mM NaCl (Fig. 4). From these results, the optimal
condition in measurement of the urchin lysozyme was determined as follows: the samples were incubated at pH 7.0, 37°C for 4 h in medium containing 100 M NaCl. The lytic activity of HEWL could not be measured in the presence of NaCl (Fig. 5) and so HEWL was unusable as a standard reagent in the measurement of urchin lysozyme. Hence, in the present study, the lytic activity of lysozyme was represented as the absorbance change at an optical density of 540 nm (OD 540 nm) before, and after the incubation. Lysozyme Activities of Acellular Coelomic Fluid and Coelomocyte Extract of S. intermedius Held at the Two Temperatures Sea urchins (n ⫽ 5) held at 25°C for 4 weeks partially had light blackish lesions at the peristomial membrane, but those (n ⫽ 6) at 11°C did not have any lessions. At these temperatures, the urchins had good feeding activity for the examined period, but we had no data on their feeding rates. In the coelomic extract, the specific lytic activity (OD 540 nm/µg protein) of the lysozyme in urchins held at 11°C appeared to be slightly higher than that of urchins at 25°C, but no distinct difference was found in activity between these temperatures (Fig. 6), while the lytic activity of this enzyme in the acellular coelomic fluid was remarkably lower than that of the coelomo-
FIG. 7. Elution patterns of protein and lysozyme fractions in coelomocyte extracts (A) and acellular coelomic fluid (B) of S. intermedius in Biogel P-30 gel chromatography. Protein (OD 595 nm, Bradfold’s method) ——; lysozyme (OD 540 nm) 哹.
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not produce enough concentrated fractions for supplying the following purification steps. Fractions precipitated by ethanol saturation. The most concentrated fractions were obtained as precipitates by 20–80% ethanol saturation in the acellular fluid and the coelomocyte extract (Table 2). The lytic activities of both samples were 3.7 times and 2.6 times that of the intact fluid and the crude extract, respectively. However, their yields were only 2.6 and 4.1%, respectively, those of the initial states. This ethanol method also could not produce a sufficient amount of purified fractions for the following purification steps.
FIG. 8. Elution patterns of lysozyme fractions of the coelomocyte exract of S. intermedius in Biogel P-30 gel chromatography. Protein (µg) – – –; lysozyme (OD 540) 哹.
cyte extract, and it was not detected in urchins held at 25°C. Purification of Sea Urchin Lysozyme Fractions salted out with ammonium sulfate. Table 1 shows the lytic activities of lysozyme fractions obtained by the saturation of various concentrations of ammonium sulfate in acellular coelomic fluid. A fraction of 50–75% (NH4)2SO4 showed the largest value in specific activity, yield, and purification. The lytic activity of this fraction was increased about three times that of the intact coelomic fluid. However, this method could
Fractions obtained by Biogel P-30 and heparin Sepharose HP gel chromatographies. At first, a single high peak fraction of lysozyme was obtained in the coelomocyte extract and the acellular coelomic fluid, respectively, in Biogel P-30 filtration (Fig. 7). However, both fractions had less protein to supply further purification procedures. At the second and subsequent trials, two peak groups of lysozyme fractions were constantly obtained in the coelomocyte extract, although there were some varieties in protein content and lytic activity. The first group (fractions 20–31) had a relatively large amount of protein, and the second group (fractions 54–77) had less protein but a high specific activity (Figs. 8 and 9, Table 3). When the first group of fractions were applied to affinity chromatography with heparin Sepharose HP, lytic activities of lysozyme were obtained in fractions 17–37, which were eluted with 0.2–0.4 mol/liter NaCl (Fig. 10). Fractions (23–36) eluted with 0.3 mol/liter NaCl showed high specific activities (Table 3). When the two samples such as the
FIG. 9. Elution patterns of lysozyme fractions in coelomocyte extract of S. intermedius in Biogel P-30 gel chromatography. protein (µg) – – –; lysozyme (OD 540) 哹.
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TABLE 3 Lysozyme Fractions Obtained by Biogel and Affinity Chromatographies in Coelomocyte Extracts of S. intermedius Fraction Coelomocyte extract (crude) Fractions of the first group (20–31) in Biogel filtration Fractions of the second group (53–77) in Biogel filtration Fractions eluted with 0.3 M NaCl in affinity chromatography
Total protein (µg/ml)
Total lytic activity (OD 540 nm ⫻ 103 )
Specific activity (OD 540 nm ⫻ 103/ µg Pr)
6469
34,056
5.3
2098
27,500
11.2
32.4
2.1
118
7541
63.8
1.8
12.0
215
18,643
86.7
3.3
16.4
fractions eluted with 0.3 mol/liter NaCl and those of the second group in the Biogel filtration were electrophoresed by SDS–PAGE, an intensely stained protein band corresponding to lysozyme was respectively obtained in each group, but many other minor protein bands were also detected (Fig. 11). The lysozyme fraction of the coelomocyte extract of S. intermedius immunologically reacted with anti-HEWL antibody (Fig. 12).
FIG. 10. Elution patterns of lysozyme fractions of the first group in Biogel filtration of S. intermedius coelomocyte extracts in affinity chromatography with a heparin Sepharose HR column. Protein (OD 280) – – –; lysozyme (OD 540) 哹.
Yield (V%, protein) 100
Purification (fold, lytic activity) 1
DISCUSSION
The lysozymes of the coelomocyte extract and acellular coelomic fluid of S. intermedius were detected as a single lytic band, respectively, in SDS–PAGE with M. lysodeikticus. The lysozyme band of the coelomocyte extract was relatively more dense and stable than that of the acellular coelomic fluid. The electrophoretic results showed that this urchin lysozyme was about 14 kDa in molecular weight, which is less than that of HEWL (14.5 kDa). Many mammalian lysozymes range from 14 to 25 kDa and those of 14–14.5 kDa are mainly contained in kidney (Audy et al., 1989), while the sea star A. rubens has a lysozyme of 15.5 kDa (Jolle`s and Jolle`s, 1975). The present results indicated that urchin lysozyme needed NaCl to display lytic activity and that its optical pH was 6.5–7.0, which was similar to that of HEWL but somewhat higher than those of marine bivalve lysozymes: 5.5 in the oyster (Crassostrea gigas) and the mussel (Mytilus edulis) (Takahashi et al., 1986), 4.6 in
FIG. 11. Electrophoretic patterns (SDS–PAGE) of purified coelomocyte extracts of S. intermedius. (1) Fractions eluted with 0.3 mol/liter NaCl in affinity chromatography, (2) fractions of the second group in Biogel P-30 gel filtration. Arrowheads indicate lysozyme bands.
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antibody of HEWL is specifically reacted with the urchin lysozyme. Therefore, the anti-HEWL antibody is useful in a quantitative analysis of the urchin lysozyme at an individual level by development of an enzyme-linked immunosobent method and in determining localization of this enzyme at the cell level by immunocytochemistry. These methods may provide further elucidation of bactericidal activity in urchins. ACKNOWLEDGMENTS
FIG. 12. Immunobloting patterns of lysozyme fractions using anti-HEWL antibody. (1) HEWL; (2) S. intermedius coelomocyte extract. Arrowheads indicate lysozyme bands.
Corbiculina leana (Mochizuki and Matsumiya, 1983), 4.5–6.0, mainly 5.0, which depends on buffer reagents in the incubation medium, in the soft-shelled clam Myaarenaria (Cheng and Rodrick, 1974). Fuji (1962) reported that the feeding rate of adult S. intermedius at 23°C decline to about a half of that at 10°C. The feeding rate of this species held at 25°C might be decreased in comparison with that at 11°C, although it was not confirmed in this study. However, no distinct difference was found in the lytic activity of urchin lysozyme between these temperatures, while this species frequently catches the spotting disease caused by bacterial infection at temperatures more than 20°C (Shimizu et al., 1995; Tajima et al., 1997). From these events, this disease of S. intermedius seems not to be caused by a decay in the lytic activity of lysozyme, and so it may be done by the predominance of a causative bacterium. The lytic activity of lysozyme was remarkably higher in the coelomocytes than the acellular coelomic fluid, and so this lytic enzyme may be produced by coelomocytes in urchins. To elucidate the origin of this enzyme and the effect of temperatures on lytic activity, however, we need further studies using many more samples with strictly controlled experimental conditions. In the present study, urchin lysozyme was purified by several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with Biogel P-30, and affinity chromatography with a heparin Sepharose HR column. The combination method of Biogel and heparin Sepharose seems to be best in obtaining a fair amount of purified enzyme. However, as the SDS–PAGE results of lysozyme-rich fractions show, the purifying procedure tried in this study was not enough to obtain a single protein band in SDS– PAGE. These facts indicate the urchin has very little lysozyme in the coelomic fluid, including coelomocytes. In the present study, however, it was confirmed that the
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Jolle`s, J., and Jolle`s, P. 1984. What’s new in lysozyme research? Always a model system, today as yesterday. Mol. Cell. Biochem. 63, 165–189. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Maes, P., and Jangoux, M. 1984. The bald-sea-urchin disease: A biopathological approach. Helgo. Meeresunters. 37, 217–224. Mo¨ck, A., and Peters, G. 1990. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. J. Fish. Biol. 37, 873–885. Mochizuki, A., and Matsumiya, M. 1983. Lysozyme activity in shellfishes. Bull. Jpn. Soc. Sci. Fish. 49, 131–135. [in Japanese] Parrinello, N., Rindone, D., and Canicatti, C. 1979. Naturally occurring hemolysins in the coelomic fluid of Holothuria polli Delle Chiaie (Echinocermata). Dev. Comp. Immunol. 3, 45–54. Roch, P. 1996. A definition of cytolytic response in invertebrates. Adv. Comp. Environ. Physiol. 23, 116–145. Sheibling, R. E., and Stephenson, R. L. 1984. Mass mortality of Strongylocentrotus droebachiensis (Echinodermata: Echinoidea) off Nova Scotia, Canada. Mar. Biol. 78, 153–164. Shimizu, M. 1992. ‘‘Studies on the Ultrastructure and Growth of Skeletons of the Test and Spines in the Sea Urchin, Strongylocentrotus intermedius.’’ Ph.d. thesis, Hokkaido University. Shimizu, M., Takaya, Y., Ohsaki, S., and Kawamata, K. 1995. Gross
and histopathological signs of the spotting disease in the sea urchin Strongylocentrotus intermedius. Fish. Sci. 61, 608–613. Smolebis, A. N., and Hartsell, S. E. 1949. The determination of lysozyme. J. Bacteriol. 58, 731–736. Stabili, L., and Canicatti, C. 1994. Antibacterial activity of the seminal plasma of Paracentrotus lividus. Can. J. Zool. 72, 1211– 1216. Stabili, L., Pagliara, P., and Roch, P. 1996. Antibacteria activity in the coelomocytes of the sea urchin Paracentrotus lividus. Comp. Biochem. Physiol. 113B, 639–644. Studnicka, M., Siwicki, A., and Ryka, B. 1986. Lysozyme level in carp (Cyprinus carpio L.). Bamidgeh 38, 22–25. Tajima, K., Hirano, T., Shimizu, M., and Ezura, Y. 1997. Isolation and pathogenicity of the causative bacterium of spotting disease of sea urchin Strongylocentrotus intermedius. Fish. Sci. 63, 249–252. Takahashi, K., Mori, K., and Nomura, T. 1986. Occurrence and characterization of lysozyme in the marine bivalves. Bull. Jpn. Soc. Sci. Fish. 52, 863–868. [in Japanese] Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polysaccharide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Wardlaw, A. C., and Unkles, S. E. 1978. Bactericidal activity of coelomic fluid from the sea urchin Echinus esculentus. J. Invertebr. Pathol. 32, 25–34.
Lytic Activity and Biochemical Properties of Lysozyme in the Coelomic Fluid of the Sea Urchin Strongylocentrotus intermedius Motohiro Shimizu,* Shin Kohno,* Hisashi Kagawa,* and Nobutoshi Ichise*,1 *Laboratory of Comparative Physiology, Department of Biology, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan Received July 11, 1997; accepted July 14, 1998
Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11° and 25°C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS–PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. r 1999 Academic Press Key Words: Strongylocentrotus intermedius; lysozyme; coelomic fluid; coelomocyte extract; immunoblotting; purification; Biogel filtration; affinity chromatography; biodefense mechanism.
INTRODUCTION
Lysozyme, known as a lytic enzyme attacking grampositive bacilli, is present in various tissues of animals and plants. This enzyme is contained in the kidney, spleen, digestive tract, and mucus of epidermis in animals (Audy et al., 1989); these organs are at risk in bacterial infection. On the other hand, stress and disease in animals affect the lytic activities of lyso-
1 Present address: Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Hokkaido 060, Japan.
0022-2011/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
zymes (Jolle`s and Jolle`s, 1984; Mo¨ck and Peters, 1990). Lysozyme may be involved in the biological defense system through not only bactericidal activity but also other procedures (Studnicka et al., 1986; Mo¨ck and Peters, 1990). We have a little information on this lytic enzyme in echinoderms, although several kinds of substances participating in biodefense mechanisms, such as lectins, hemolysins, aggultinins, and others, have been identified (Wardlaw and Unkles, 1978; Parrinello et al., 1979; Canicatti, 1987, 1989, 1991, 1992; Canicatti and Ciulla, 1987; Azumi et al., 1991; Stabili et al., 1996; Roch, 1996). Jolle`s and Jolle`s (1975) studied the biochemical properties of lysozyme in the sea star Asterias rubens. In the sea cucumber Holothuria polii, lysozyme-like lytic activity was also found in coelomocytes (Canicatti et al., 1989). Canicatti and D’Ancona (1990) also confirmed lysozyme in the mucus of the sea star Marthasterias glacialis. In the sea urchin Paracentrotus lividus, Stabili and Canicatti (1994) identified this enzyme as one of the antibacterial factors in the seminal plasma. However, we have less information on this lytic enzyme in the coelomic fluid of sea urchins. Recently, ‘‘spotting disease’’ or ‘‘spine-shedding disease’’ has been found in sea urchins living on some coasts and in breeding stations in Japan (Hamaguti et al., 1993; Shimizu et al., 1995; Tajima et al., 1997). Similar urchin diseases have been reported in urchins living in several ocean coasts of Europe, Canada, and other areas (Jangoux, 1984; Maes and Jangoux, 1984; Scheibling and Stephenson, 1984). These diseases cause high motality rates. In the Akauni (red sea urchin) Pseudocentrotus depressus affected by the spineshedding disease, a bacillus, Frexbactor sp., was identified as a pathogen (Hamaguti et al., 1993). A similar bacillus was also found in spotting disease of Strongylocentrotus intermedius and is supposed to be a pathogen of the disease (Shimizu et al., 1995; Tajima et al., 1997). The latter species lives in northern Japan and falls to the disease in high temperature seasons, but the former type, living in southern Japan, does so in low temperature seasons. The bacillus infection and unpro-
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pitious temperatures for the urchins may affect their biodefense system. Therefore, if they have lysozyme, its lytic activity may respond to these environmental factors. In the present study, we identified lysozyme in the coelomic fluid, including coelomocytes, in S. intermedius, examined its lytic activity in urchins held at the two different water temperatures, and then clarified some biochemical properties of this enzyme. Furthermore, we tried to purify the urchin lysozyme and identify the immunological resemblance between this enzyme and hen egg white lysozyme by immunoblotting. MATERIALS AND METHODS
Experimental Animals About 150 specimens (50 g and 56 mm in mean body weight and test diameter, respectively) of S. intermedius were used in this study. They were collected from the tidal zone of the coast near Hakodate in spring and summer (environmental seawater temperatures, about 5–20°C) and reared in the Usujiri Experimental Marine Station of Hokkaido University near Hakodate until use. Ten individuals each were held at the two different water temperatures of 11° and 25°C, respectively, for 4 weeks in aquaria (30 ⫻ 20 ⫻ 30 cm) equipped with a circulating filter system. The seawater used was filtered with a mesh of 1 µm pore size (Organo PF Model III). Wakame, Undaria, fresh or desalted from salted ones, was given as food every 2 days. Preparation of the Samples for Lysozyme Assay Coelomic fluid was withdrawn using plastic spoids from the coelom by puncture of the peristomial membrane into a 10-ml plastic test tube and then centrifuged at 3000 rpm for 10 min to precipitate coelomocytes. The supernatants of three to five tubes were pooled and filtered with a paper filter (Toyo No. 6), dialyzed against 0.67 M phosphate buffer (pH 6.8) for 6 h, and lyophilized as the sample of acellular coelomic fluid. The precipitates of those tubes were pooled and homogenized with 3–5 ml phosphate buffer and centrifuged at 15,000 rpm for 10 min to separate the supernatant as the sample of coelomocyte extract. These samples were stored at ⫺20°C until use.
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Fifty microliters of the specimens was mixed with 3 ml of the medium. At first, the samples were incubated at 11°, 20°, and 37°C for 15, 90, and 240 min to determine the optimal incubation temperature and time of its lytic activity. Lytic activity was detected by measuring the absorbancy at 540 nm. Based on the results of these preliminary experiments, the following incubations were done at 37°C for 4 h. Optimal pH in lytic activity was determined at various pHs using medium containing 100 mM NaCl and optimal NaCl concentration done at pH 7.0 using media containing NaCl at various concentrations (0–200 mM). To examine the availability of hen egg white lysozyme (HEWL) as the standard substance in the measurement of the urchin lysozyme, HEWL lytic activity was preliminarily measured using media containing NaCl at various concentrations (0, 100, and 200 mM), for 15 min at pH 7.0 and 37°C. Electrophoretic Examination of Lysozyme Fraction Lysozyme fraction was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) by the method of Audy et al. (1989). The gel (15%) contained 0.4% Micrococcus lysodeikticus. Each sample (lyophilized coelomic fluid, coelomocyte extract, and HEWL) was mixed with the same volume of a sample buffer (0.01% Tris–HCl, pH 6.8) containing 0.1% SDS and 2% sucrose and boiled for 2 min. It was electrophoretically fractionated by SDS–PAGE at 30 mA in accordance with the method of Laemmli (1970). After electrophoresis, the gels were incubated with an activating buffer (50 mM sodium phosphate, 100 mM NaCl, 1% Triton X-100, pH 6.5) in a sealed well at 37°C for 4 h. Lytic zones with lysozymes were detected under a black background: the zone was observed to be transparent. Purification of Sea Urchin Lyzozyme About 120 individuals were used for purification of the lysozyme of the coleomic fluid and coelomocytes.
Measurement of Lysozyme Activity Lytic activity of lysozyme was measured spectrophotometrically by the method of Smolebis and Hartsell (1949). Twenty-four micrograms of dried M. lysodeikticus was mixed with 100 ml 0.67 M phosphate buffer, kept for 2 days, and then centrifuged. The supernatant was used as the medium for lysozyme measurement.
FIG. 1. Lysozyme fractions in SDS–PAGE of the acellular coelomic fluid and coelomocyte extract of S. intermedius. 1, hen egg white lysozyme (HEWL); 2–5, coelomocyte extract; 6, 7, acellular coelomic fluid.
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FIG. 2. Effects of incubation temperature and time on the lytic activity of lysozyme in coelomocyte extracts of S. intemedius.
The lyophilized acellular coelomic fluid and the coelomic extract were obtained by the method described before. Precipitation by saturating ammonium sulfate or ethanol. At first, the samples were salted out with ammonium sulfate at 35, 50, 75, and 90% saturation. The precipitated proteins in each saturated state were collected by centrifugation (10,000g, 20 min, 0°C) and dissolved to 0.05 mol/liter So¨rensen phosphate buffer (pH 6.8). As the secondary, the samples were brought to 20% saturation with ethanol, stirred for 1 h, and then centrifuged at 10,000g for 20 min. The supernatant was then adjusted to 80% saturation with additional etha-
FIG. 3. Effect of pH on the lytic activity of lysozyme in coelomocyte extracts of S. intermedius.
FIG. 4. Effect of NaCl concentrations on the lytic activity of lysozyme in coelmocyte extracts of S. intermedius.
nol and stirred for 1 h, and the precipitated proteins were collected by centrifugation as above. Gel filtration with Biogel P-30. The supernatant at 20% ethanol saturation was layered on a Biogel P-30 column (3 ⫻ 70 cm) equilibrated with 0.05 mol/liter So¨rensen phosphate buffer (pH 6.0), eluted with the same buffer at 0.4 ml/s, and collected at 4 ml in each tube. The combination method of gel filtration (Biogel P-30) and affinity chromatography (heparin Sepharose HP). The coelomocyte extracts obtained from 23 individuals were dialyzed against 0.05 mol/liter phosphate buffer (pH 6.0) and then applied to the Biogel P-30 column equilibrated with the same buffer. They were eluted with the same buffer at one drop/7 s, and 4 ml was collected in each tube. The fractions having lysozymal lytic activity were pooled and applied to the heparin
FIG. 5. Effect of NaCl concentrations on the lytic activity of hen egg whate lysozyme.
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adjuvant (Wako Pure Reagent Co. Japan) to form an emulsion. The emulsion was subcutaneusly injected into a rabbit six times once a week. When a sufficient formation of antibody was confirmed by using a little blood collected from an epiotic vein, whole blood was obtained by puncturing the heart. The blood was kept for 1 h at 37°C and overnight in a refrigerator (4°C) and then centrifuged at 3000 rpm for 20 min at 4°C to obtain the supernatant as anti-HEWL rabbit serum. The antiserum was kept ⫺80°C until use.
FIG. 6. Lytic activities of lysozyme in acellular coelomic fluid and coelomocyte extracts of S. intermedius acclimated to different water temperatures (11° and 25°C). The activity of acellular coelomic fluid was not determined at 25°C. The result is expressed as the mean ⫾ SE (bar).
Sepharose HP column equilibrated with the phosphate buffer. The protein fractions were eluted stepwise with the same buffer containing 0.1–1 mol/liter NaCl at a drop/4 s, and 2 ml each was collected. The peak fractions were pooled and applied to SDS–PAGE to determine the purification state of the lysozyme fraction. The lysozymal lytic activity of each fraction in all purification steps was measured by absorbancy at 540 nm and the protein content was done by the method of Bradford (1976) or absorbancy at 280 nm, and then a specific activity (OD 540 nm/µg protein), yield (volume %, protein), and purification (fold, lytic activy) were calculated in each fraction. Analysis of Immunological Similarity between Sea Urchin Lysozyme and HEWL Formation of anti-serum against HEWL. Five hundred micrograms of HEWL (Sigma) was dissolved in 1 ml of 0.05 mol/liter phosphate buffer (pH 6.0) and then mixed with the same volume of Freund’s complete
Immunoelectrophoresis. Immunoelectrophoresis of the coelomocyte extracts was performed by the method of Grabar and Williams (1953). The gel was made of Veronal acetate buffer (10 mM Veronal, 50 mM sodium Veronal; pH 8.6) containing 1% agarose. The extracts were electrophoresed at a constant current, 3.6 mA/cm, until a running front reached 3 cm long from the starting point. When the electrophoresis was finished, a groove was made on the gel parallel to the running sample. One hundred twenty microliters of the antiserum was applied to the groove and kept overnight. After being washed with a physiological solution and distilled water, the gel was dried and then stained with Coomassie brilliant blue (CBB) staining solution (0.1% CBB R-250 in a mixture of ethanol:acetic acid:distilled water 1:1:1). Immunoblotting. The coelomocyte extract and HEWL were respectively dissolved in 0.05 mol/liter phosphate buffer (pH 6.0) to 5 mg/ml final concentration and then applied to SDS–PAGE in accordance with the methods of Laemmli (1970). After electrophoresis, the fractionated protein bands were transferred to polyvinyldendifuruolid membrane (PVDF, Millipore) by the method of Towbin et al. (1979). They were blocked for 1 h with 5% skim milk with Tris-buffered sodium chloride solution (TBS: 20 mM Tris, 500 mM NaCl, pH 7.5) and then reacted with diluted primary antiserum (anti-HEWL rabbit serum) at 37°C for 1 h. After the PVDF was washed briefly with distilled water, twice 10 min each with 0.05% Tween 20 in TBS (TTBS), and 10 min with TBS, it was treated with the secondary antibody (horseradish peroxidase-labeled
TABLE 1 Protein Contents and Lytic Activities of Lysozyme Fractions Salted out with Ammonium Sulfate in Acellular Coelomic Fluid of S. intermedius Fraction Acellular coelomic fluid Ammonium sulfate saturation 0–35% 35–50% 50–75% 75–90%
Total protein (µg)
Total lytic activity (OD 540 nm ⫻ 103 )
Specific activity (OD 540 nm ⫻ 103/ µg Pr)
5900
5380
0.912
500 590 670 190
130 720 1650 340
0.259 1.221 2.466 1.813
Yield (V%, protein) 100 8.4 10.6 11.3 3.2
Purification (fold, lytic activity) 1 0.28 1.34 2.70 1.99
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TABLE 2 Lysozyme Fractions Isolated by Ethanol Saturation in Acellular Coelomic Fluid and Coelomocyte Extracts of S. intermedius Fraction
Total protein (µg)
Total lytic activity (OD 540 nm ⫻ 103 )
Specific activity (OD 540 nm ⫻ 103/ µg Pr)
Yield (V%, protein)
Purification (fold, lytic activity)
Acellular coelomic fluid (crude) 20–80% ethanol sediment Coelomocyte extract (crude) 20–80% ethanol sediment
135,000 2,870 71,880 2,920
278,400 2,200 80,940 8,530
2.1 7.6 1.13 2.92
100 2.6 100 4.1
1.0 3.6 1.0 2.6
anti-rabbit IgG goat serum, Sigma A-9169) for 2 h at room temperature. Consecutively, the membrane was washed twice with TTBS (10 min each) and TBS (10 min) and colored with a mixture of 0.06% 4-chloro-1naphtol, 0.01% hydrogen oxide, and 20% methanol. The membrane was dried after wash with distilled water. RESULTS
Lysozyme Fractions of Acellular Coelomic Fluid and Coelomocytes of S. intermedius In SDS–PAGE, a clear lytic zone was found in the coelomocyte extract (Fig. 1). The colomic fluid showed a faint zone at the same position. Both lysozyme zones were situated at about 14 kDa, being a little smaller than HEWL (14.5 kDa) in molecular weight. Identification of an Optimum Method for Measuring the Lytic Activity of Urchin Lysozyme Lysozyme of the coelomocyte extract grew in its lytic activity as the increase in the examined incubation temperature and time; the highest activity was obtained at 37°C for 4 h (Fig. 2). The lytic activities were high at pH 6.5–7.0 and decreased in ranges lower than pH 5 and higher than pH 8 (Fig. 3). Its activity was maximum when the incubation medium contained 100 mM NaCl (Fig. 4). From these results, the optimal
condition in measurement of the urchin lysozyme was determined as follows: the samples were incubated at pH 7.0, 37°C for 4 h in medium containing 100 M NaCl. The lytic activity of HEWL could not be measured in the presence of NaCl (Fig. 5) and so HEWL was unusable as a standard reagent in the measurement of urchin lysozyme. Hence, in the present study, the lytic activity of lysozyme was represented as the absorbance change at an optical density of 540 nm (OD 540 nm) before, and after the incubation. Lysozyme Activities of Acellular Coelomic Fluid and Coelomocyte Extract of S. intermedius Held at the Two Temperatures Sea urchins (n ⫽ 5) held at 25°C for 4 weeks partially had light blackish lesions at the peristomial membrane, but those (n ⫽ 6) at 11°C did not have any lessions. At these temperatures, the urchins had good feeding activity for the examined period, but we had no data on their feeding rates. In the coelomic extract, the specific lytic activity (OD 540 nm/µg protein) of the lysozyme in urchins held at 11°C appeared to be slightly higher than that of urchins at 25°C, but no distinct difference was found in activity between these temperatures (Fig. 6), while the lytic activity of this enzyme in the acellular coelomic fluid was remarkably lower than that of the coelomo-
FIG. 7. Elution patterns of protein and lysozyme fractions in coelomocyte extracts (A) and acellular coelomic fluid (B) of S. intermedius in Biogel P-30 gel chromatography. Protein (OD 595 nm, Bradfold’s method) ——; lysozyme (OD 540 nm) 哹.
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not produce enough concentrated fractions for supplying the following purification steps. Fractions precipitated by ethanol saturation. The most concentrated fractions were obtained as precipitates by 20–80% ethanol saturation in the acellular fluid and the coelomocyte extract (Table 2). The lytic activities of both samples were 3.7 times and 2.6 times that of the intact fluid and the crude extract, respectively. However, their yields were only 2.6 and 4.1%, respectively, those of the initial states. This ethanol method also could not produce a sufficient amount of purified fractions for the following purification steps.
FIG. 8. Elution patterns of lysozyme fractions of the coelomocyte exract of S. intermedius in Biogel P-30 gel chromatography. Protein (µg) – – –; lysozyme (OD 540) 哹.
cyte extract, and it was not detected in urchins held at 25°C. Purification of Sea Urchin Lysozyme Fractions salted out with ammonium sulfate. Table 1 shows the lytic activities of lysozyme fractions obtained by the saturation of various concentrations of ammonium sulfate in acellular coelomic fluid. A fraction of 50–75% (NH4)2SO4 showed the largest value in specific activity, yield, and purification. The lytic activity of this fraction was increased about three times that of the intact coelomic fluid. However, this method could
Fractions obtained by Biogel P-30 and heparin Sepharose HP gel chromatographies. At first, a single high peak fraction of lysozyme was obtained in the coelomocyte extract and the acellular coelomic fluid, respectively, in Biogel P-30 filtration (Fig. 7). However, both fractions had less protein to supply further purification procedures. At the second and subsequent trials, two peak groups of lysozyme fractions were constantly obtained in the coelomocyte extract, although there were some varieties in protein content and lytic activity. The first group (fractions 20–31) had a relatively large amount of protein, and the second group (fractions 54–77) had less protein but a high specific activity (Figs. 8 and 9, Table 3). When the first group of fractions were applied to affinity chromatography with heparin Sepharose HP, lytic activities of lysozyme were obtained in fractions 17–37, which were eluted with 0.2–0.4 mol/liter NaCl (Fig. 10). Fractions (23–36) eluted with 0.3 mol/liter NaCl showed high specific activities (Table 3). When the two samples such as the
FIG. 9. Elution patterns of lysozyme fractions in coelomocyte extract of S. intermedius in Biogel P-30 gel chromatography. protein (µg) – – –; lysozyme (OD 540) 哹.
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TABLE 3 Lysozyme Fractions Obtained by Biogel and Affinity Chromatographies in Coelomocyte Extracts of S. intermedius Fraction Coelomocyte extract (crude) Fractions of the first group (20–31) in Biogel filtration Fractions of the second group (53–77) in Biogel filtration Fractions eluted with 0.3 M NaCl in affinity chromatography
Total protein (µg/ml)
Total lytic activity (OD 540 nm ⫻ 103 )
Specific activity (OD 540 nm ⫻ 103/ µg Pr)
6469
34,056
5.3
2098
27,500
11.2
32.4
2.1
118
7541
63.8
1.8
12.0
215
18,643
86.7
3.3
16.4
fractions eluted with 0.3 mol/liter NaCl and those of the second group in the Biogel filtration were electrophoresed by SDS–PAGE, an intensely stained protein band corresponding to lysozyme was respectively obtained in each group, but many other minor protein bands were also detected (Fig. 11). The lysozyme fraction of the coelomocyte extract of S. intermedius immunologically reacted with anti-HEWL antibody (Fig. 12).
FIG. 10. Elution patterns of lysozyme fractions of the first group in Biogel filtration of S. intermedius coelomocyte extracts in affinity chromatography with a heparin Sepharose HR column. Protein (OD 280) – – –; lysozyme (OD 540) 哹.
Yield (V%, protein) 100
Purification (fold, lytic activity) 1
DISCUSSION
The lysozymes of the coelomocyte extract and acellular coelomic fluid of S. intermedius were detected as a single lytic band, respectively, in SDS–PAGE with M. lysodeikticus. The lysozyme band of the coelomocyte extract was relatively more dense and stable than that of the acellular coelomic fluid. The electrophoretic results showed that this urchin lysozyme was about 14 kDa in molecular weight, which is less than that of HEWL (14.5 kDa). Many mammalian lysozymes range from 14 to 25 kDa and those of 14–14.5 kDa are mainly contained in kidney (Audy et al., 1989), while the sea star A. rubens has a lysozyme of 15.5 kDa (Jolle`s and Jolle`s, 1975). The present results indicated that urchin lysozyme needed NaCl to display lytic activity and that its optical pH was 6.5–7.0, which was similar to that of HEWL but somewhat higher than those of marine bivalve lysozymes: 5.5 in the oyster (Crassostrea gigas) and the mussel (Mytilus edulis) (Takahashi et al., 1986), 4.6 in
FIG. 11. Electrophoretic patterns (SDS–PAGE) of purified coelomocyte extracts of S. intermedius. (1) Fractions eluted with 0.3 mol/liter NaCl in affinity chromatography, (2) fractions of the second group in Biogel P-30 gel filtration. Arrowheads indicate lysozyme bands.
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antibody of HEWL is specifically reacted with the urchin lysozyme. Therefore, the anti-HEWL antibody is useful in a quantitative analysis of the urchin lysozyme at an individual level by development of an enzyme-linked immunosobent method and in determining localization of this enzyme at the cell level by immunocytochemistry. These methods may provide further elucidation of bactericidal activity in urchins. ACKNOWLEDGMENTS
FIG. 12. Immunobloting patterns of lysozyme fractions using anti-HEWL antibody. (1) HEWL; (2) S. intermedius coelomocyte extract. Arrowheads indicate lysozyme bands.
Corbiculina leana (Mochizuki and Matsumiya, 1983), 4.5–6.0, mainly 5.0, which depends on buffer reagents in the incubation medium, in the soft-shelled clam Myaarenaria (Cheng and Rodrick, 1974). Fuji (1962) reported that the feeding rate of adult S. intermedius at 23°C decline to about a half of that at 10°C. The feeding rate of this species held at 25°C might be decreased in comparison with that at 11°C, although it was not confirmed in this study. However, no distinct difference was found in the lytic activity of urchin lysozyme between these temperatures, while this species frequently catches the spotting disease caused by bacterial infection at temperatures more than 20°C (Shimizu et al., 1995; Tajima et al., 1997). From these events, this disease of S. intermedius seems not to be caused by a decay in the lytic activity of lysozyme, and so it may be done by the predominance of a causative bacterium. The lytic activity of lysozyme was remarkably higher in the coelomocytes than the acellular coelomic fluid, and so this lytic enzyme may be produced by coelomocytes in urchins. To elucidate the origin of this enzyme and the effect of temperatures on lytic activity, however, we need further studies using many more samples with strictly controlled experimental conditions. In the present study, urchin lysozyme was purified by several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with Biogel P-30, and affinity chromatography with a heparin Sepharose HR column. The combination method of Biogel and heparin Sepharose seems to be best in obtaining a fair amount of purified enzyme. However, as the SDS–PAGE results of lysozyme-rich fractions show, the purifying procedure tried in this study was not enough to obtain a single protein band in SDS– PAGE. These facts indicate the urchin has very little lysozyme in the coelomic fluid, including coelomocytes. In the present study, however, it was confirmed that the
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