Purification and properties of phospholipases A2 from the crown-of-thorns starfish (Acanthaster planci) venom

PII: S0041-0101(97)00085-8

Toxicon Vol. 36, No. 4, pp. 589±599, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0041-0101/98 $19.00 + 0.00

PURIFICATION AND PROPERTIES OF PHOSPHOLIPASES A2 FROM THE CROWN-OFTHORNS STARFISH (ACANTHASTER PLANCI) VENOM K.AZUO SHIOMI,* AKEMI KAZAMA, KUNIYOSHI SHIMAKURA and YUJI NAGASHIMA Department of Food Science and Technology, Tokyo University of Fisheries, Konan-4, Minato-ku, Tokyo 108, Japan (Received 9 May 1997; accepted 9 June 1997)

K. Shiomi, A. Kazama, K. Shimakura and Y. Nagashima. Puri®cation and properties of phospholipases A2 from the crown-of-thorns star®sh (Acanthaster planci) venom. Toxicon 36, 589±599, 1997. Ð Two phospholipases A
INTRODUCTION

The crown-of-thorns star®sh Acanthaster planci has a number of venomous spines on the body surface. Contact with the spines in¯icts noxious symptoms such as severe pain, redness, swelling and protracted vomiting. Taira et al. (1975) ®rst reported that the crude toxin extracted from the spines exhibits mouse lethality and hemolytic ac* Author to whom correspondence should be addressed. 589

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tivity. Subsequently, the following diverse biological activities were also detected in the crude toxin: myonecrotic activity, hemorrhagic activity, capillary-permeabilityincreasing activity, edema-forming activity, phospholipase A2 (PLA2) activity (Shiomi et al., 1985), histamine-releasing activity from mast cells (Shiomi et al., 1989), cardiovascular actions (Yara et al., 1992; Shiroma et al., 1994) and anticoagulant activity (Karasudani et al., 1996). A lethal factor of glycoproteinic nature has already been puri®ed (Shiomi et al., 1988) and shown to be potently hepatotoxic (Shiomi et al., 1990; Shiomi et al., 1991). Very recently, an anticoagulant factor of peptidic nature has also been isolated and characterized (Karasudani et al., 1996). However, both lethal and anticoagulant factors are not responsible for most of the biological activities detected in the crude toxin and hence the symptoms induced by A. planci stings are not fully understood. PLA2s, enzymes hydrolysing the sn-2 ester bond of phospholipids, are abundant in venoms of snakes and insects as well as in mammalian pancreatic juices. It is well known that venom PLA2s, especially snake venom PLA2s, are deeply associated with various pathological symptoms in poisonings. This may be the case with the PLA2s detected in the A. planci venom. In this study, therefore, we attempted to purify PLA2s from the A. planci venom and clarify their properties. MATERIALS AND METHODS Star®sh Specimens of A. planci were captured along the coasts of Kuroshima Island, Okinawa Prefecture, in October 1989. They were frozen after capture, shipped to our laboratory and kept at ÿ208C until use. Isolation procedure Spines (227 g) were collected from three specimens of A. planci and extracted twice with 2 volumes of 0.01 M phosphate bu€er (pH 7.0). The extract (crude toxin) was dialyzed thoroughly against the same bu€er and the inner solution was applied to a CM-cellulose column (2  48 cm; Brown, Berlin, U.S.A.). The column was eluted stepwise with 0.01 M phosphate bu€er (pH 7.0) and 1.0 M NaCl in the same bu€er and fractions of 15 ml were collected at a ¯ow rate of about 55 ml/h. Active fractions were pooled and added slowly with solid ammonium sulfate to give a ®nal concentration of 80% saturation. The precipitate formed was collected by centrifugation and dissolved in 100 ml of 0.5 M ammonium sulfate in 0.01 M phosphate bu€er (pH 7.0). After removal of insoluble materials by centrifugation, the solution was put onto a Phenyl Sepharose CL-4B column (2  40 cm; Pharmacia, Uppsala, Sweden). The column was eluted successively with 0.5 M ammonium sulfate in 0.01 M phosphate bu€er (pH 7.0), 0.01 M phosphate bu€er (pH 7.0) and 25% ethylene glycol; fractions of 15 ml were collected at a ¯ow rate of about 60 ml/h. PLA2 activity was detected in two fractions (PLA2-I and II fractions; see Fig. 1). Both fractions were separately concentrated to about 5 ml by ultra®ltration using a mini module NM-3 (Asahi Kasei, Tokyo, Japan). The concentrate of the PLA2-I fraction was chromatographed on a Sephacryl S-200 column (2  50 cm; Pharmacia) with 0.15 M NaCl in 0.01 M phosphate bu€er (pH 7.0). Fractions of 3 ml were collected at a ¯ow rate of about 20 ml/h. PLA2-containing fractions were pooled and subjected to reverse-phase HPLC on a TSKgel Phenyl 5PW-RP column (0.46  7.5 cm; Tosoh, Tokyo, Japan). After washing with 0.1% tri¯uoroacetic acid (TFA), the column was eluted at a ¯ow rate of 0.5 ml/min by a gradient of acetonitrile (see Fig. 2(B) for details) in 0.1% TFA. Thus, one PLA2 component (AP-PLA2-I) was isolated. On the other hand, the concentrate of the PLA2-II fraction obtained by hydrophobic chromatography on Phenyl Sepharose CL-4B was ®rst applied to gel ®ltration FPLC on a Superose 12 HR10/30 column (1  30 cm; Pharmacia), which was eluted with 0.15 M NaCl in 0.01 M phosphate bu€er (pH 7.0) at a ¯ow rate of 0.5 ml/min; fractions of 0.5 ml were manually collected. Active fractions were combined and ®nally subjected to reverse-phase HPLC on a TSKgel ODS120T column (0.46  25 cm; Tosoh) to isolate AP-PLA2-II. Elution of the column was achieved at a ¯ow rate of 1 ml/min by a gradient of acetonitrile (see Fig. 3(B) for details) in 0.1% TFA. Assay of PLA2 activity and positional speci®city Throughout the puri®cation procedure, PLA2 activity was followed by measuring the clearing of egg yolk suspension, essentially according to the method of Marinetti (1965). To 0.1 ml of sample solution was added

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Fig. 1. Separation of PLA2s by hydrophobic chromatography on Phenyl Sepharose CL-4B. The PLA2 fraction obtained by CM-cellulose column chromatography was applied to a Phenyl Sepharose CL-4B column (2  40 cm). After washing with 0.5 M ammonium sulfate in 0.01 M phosphate bu€er (pH 7.0), the column was eluted successively with 0.01 M phosphate bu€er (pH 7.0) and 25% ethylene glycol, as indicated by arrows. Fractions of 15 ml were collected at a ¯ow rate of about 60 ml/h. Bars represent active fractions (PLA2-I and II fractions).

1.5 ml of egg yolk suspension which was made in 0.1 M Tris±HCl bu€er (pH 8.0) at a concentration of 2 mg egg yolk/ml. The absorbance at 900 nm of the mixture was estimated for initial 5 min. One enzyme unit was de®ned as the activity causing the decrease of 0.01 in absorbance/min. In order to examine enzymatic properties (optimum pH and e€ects of divalent cations) of the puri®ed PLA2s, the activity was assayed using egg yolk phosphatidylcholine (PC) as a substrate by the method of Bhat and Gowda (1989). Fatty acids released from PC were determined based on the calibration curve prepared using linoleic acid as a standard. For the estimation of the optimum pH, the enzyme reaction was performed in the following 0.05 M bu€ers without divalent cations: acetate bu€er (pH 5.0), phosphate bu€er (pH 6.0 or 7.0), Tris±HCl bu€er (pH 7.5 or 8.0) and glycine±NaOH bu€er (pH 9.0 or 10.0). E€ects of divalent cations on the PLA2 activity were evaluated from the enzyme reaction in 0.05 M Tris±HCl bu€er (pH 7.5) with 5 mM Ca2+, Cu2+, Fe2+, Mg2+, Mn2+ or Zn2+ or without divalent cations. The positional speci®city of the puri®ed enzymes was examined by a slight modi®cation of the method of Vishwanath et al. (1987). In this study, synthetic PC having a fatty acid labeled with 4-nitrobenz-2-oxa-1,3-diazole at the sn-2 position (Funakoshi, Tokyo, Japan) was used as a ¯uorescent substrate. After incubation of the reaction mixture (containing 1 mg of enzyme and 2000 nmol of ¯uorescent PC) at 378C for 30 min, the products formed were analyzed by TLC on a silica gel plate with chloroform±methanol±acetic acid (20:9:4, v/v/v). Fluorescent spots were visualized under UV light. Hemolytic activity The hemolytic activity of the puri®ed PLA2s against sheep erythrocytes was examined with or without egg yolk PC. PC was dissolved in methanol and the enzymes in bu€ered saline (0.15 M NaCl and 5 mM Ca2+ in 0.01 M Tris±HCl bu€er, pH 7.5). To the sheep blood was added about 30 volumes of bu€ered saline and the mixture was centrifuged. The erythrocyte pellet was washed twice with bu€ered saline and a 2% erythrocyte suspension was prepared in bu€ered saline. The reaction mixture was composed of 2.9 ml of enzyme solution (containing 0.1 or 1.0 mg of enzyme), 0.1 ml of PC solution (containing 100 nmol of PC) or methanol and 1.0 ml of a 2% erythrocyte suspension. Following incubation at 378C for 30 min, the mixture was centrifuged brie¯y. The percentage of hemolysis was estimated from the absorbance at 542 nm of the hemoglobin released.

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Fig. 2. Puri®cation of AP-PLA2-I by gel ®ltration on Sephacryl S-200 (A) and reverse-phase HPLC on TSKgel Phenyl 5PW-RP (B). (A) The PLA2-I fraction obtained by hydrophobic chromatography (Fig. 1) was applied to gel ®ltration on a Sephacryl S-200 column (2  50 cm), which was eluted with 0.15 M NaCl in 0.01 M phosphate bu€er (pH 7.0) at a ¯ow rate of about 20 ml/h. Fractions of 3 ml were collected. A bar represents fractions with PLA2 activity. (B) The active fraction obtained by gel ®ltration was subjected to reverse-phase HPLC on a TSKgel Phenyl 5PW-RP column (0.46  7.5 cm). After washing with 0.1% TFA, the column was eluted at a ¯ow rate of 0.5 ml/min by a gradient of acetonitrile in 0.1% TFA. An arrow represents the peak containing AP-PLA2-I. Protein determination Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.

Molecular mass determination The molecular masses of the puri®ed enzymes were determined by either gel ®ltration HPLC or sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel ®ltration HPLC was performed on a TSKgel G3000SW column (0.75  30 cm; Tosoh, Tokyo, Japan) with 0.15 M NaCl in 0.01 M phosphate bu€er (pH 7.0). The column was eluted at a ¯ow rate of 0.5 ml/min and the eluate was continuously monitored at 280 nm with a UV detector. The reference proteins used were bovine serum albumin (67 kDa), ovalbumin (43 kDa) and myoglobin (17 kDa). In SDS-PAGE, running, staining and destaining were all carried out on a PhastSystem apparatus (Pharmacia, Uppsala, Sweden), according to the programs recommended by the manufacturer. Ready-made slab gels (PhastGel Gradient 10±15), ready-made bu€er strips (PhastGel SDS Bu€er Strips) and a low molecular weight calibration kit containing phosphorylase B (94 kDa), bovine serum albumin, ovalbumin, carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa) were purchased from Pharmacia. Prior to electrophoresis, each puri®ed enzyme was dissolved in 0.01 M Tris±HCl bu€er (pH 6.8) containing 1% SDS, 1% 2-mercaptoethanol and 20% glycerin and heated in boiling water for 10 min to denature.

Amino acid analysis Each puri®ed enzyme (30 mg) was hydrolysed with 50 ml of 4 M methanesulfonic acid containing 0.2% 3-(2aminoethyl) indole in an evacuated tube at 1158C for 24 h. After hydrolysis, the solution was neutralized with 50 ml of 3.5 M NaOH and then diluted with 500 ml of 0.2 M citrate bu€er (pH 2.2). A 500 ml portion of the dilution was applied to an amino acid analyzer (Atto MLC-703).

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Fig. 3. Puri®cation of AP-PLA2-II by gel ®ltration FPLC on Superose 12 (A) and reverse-phase HPLC on TSKgel ODS-120T (B). (A) The PLA2-II fraction obtained by hydrophobic chromatography (Fig. 1) was applied to gel ®ltration FPLC on a Superose 12 column (1  30 cm), which was eluted with 0.15 M NaCl in 0.01 M phosphate bu€er (pH 7.0) at a ¯ow rate of 0.5 ml/min. Fractions of 0.5 ml were collected. A bar indicates fractions with PLA2 activity. (B) The active fraction obtained by gel ®ltration FPLC was subjected to reverse-phase HPLC on a TSKgel ODS-120T column (0.46  25 cm). After washing with 0.1% TFA, the column was eluted at a ¯ow rate of 1 ml/min by a gradient of acetonitrile in 0.1% TFA. An arrow represents the peak containing AP-PLA2-II. Sequence analysis The N-terminal sequences of the puri®ed enzymes were determined by the automated Edman degradation method using a gas-phase protein sequencer (Beckman LF-3400D TriCart with high sensitivity chemistry).

RESULTS

Puri®cation of PLA2s When the crude toxin was applied to a CM-cellulose column, PLA2s were passed through the column, suggesting that they are neutral or acidic proteins. Although great amounts of contaminated proteins were also passed through the column (see Table 1), this step was e€ective to separate PLA2s from the lethal factor which was retained on the column as reported previously (Shiomi et al., 1988). In hydrophobic chromatography on Phenyl Sepharose CL-4B, PLA2s appeared in two fractions, PLA2-I fraction eluted with 0.01 M phosphate bu€er (pH 7.0) and PLA2-II fraction eluted with 25% ethylene glycol (Fig. 1). AP-PLA2-I contained in the PLA2-I fraction was further puri®ed by gel ®ltration on Sephacryl S-200 and reverse-phase HPLC on TSKgel Phenyl 5PW-RP (Fig. 2) and AP-PLA2-II in the PLA2-II fraction by gel ®ltration FPLC on Superose 12 and reverse-phase HPLC on TSKgel ODS-120T (Fig. 3). Both AP-PLA2-I and II were eluted in a symmetrical peak in the last step of puri®cation (reverse-phase HPLC). Thus, 3.4 mg of AP-PLA2-I and 3.5 mg of AP-PLA2-II were obtained from

594

K. SHIOMI et al. Table 1. Summary of PLA2 puri®cation from A. planci

Step Crude toxin CM-cellulose Phenyl Sepharose CL-4B PLA2-I fraction PLA2-II fraction AP-PLA2-I Sephacryl S-200 TSKgel Phenyl 5PW-RP AP-PLA2-II Superose 12 TSKgel ODS-120T

Protein (mg)

Speci®c PLA2 activity (units/mg)

Total PLA2 activity (units)

Recovery in PLA2 activity (%)

4820 4420

830 860

4 000 000 3 800 000

100 95

190 111

2470 1550

469 000 173 000

12 4.3

31 3.4

2780 7830

87 000 26 700

2.2 0.7

6.3 3.5

15 800 16 600

100 000 57 400

2.5 1.4

Puri®cation was performed using 227 g of spines collected from three specimens of A. planci. PLA2 activity was assayed by measuring the clearing of egg yolk suspension, essentially according to the method of Marinetti (1965). One enzyme unit was de®ned as the activity causing the decrease of 0.01 in absorbance at 900 nm of egg yolk suspension per min.

227 g of spines, although the total recovery (AP-PLA2-I + II) in activity was only 2.1% (Table 1). AP-PLA2-II (16,600 units/mg) was about two-fold higher in enzyme activity than AP-PLA2-I (7830 units/mg). Properties of the puri®ed PLA2s In the absence of PC, no hemolysis of sheep erythrocytes was displayed by 1.0 mg of either AP-PLA2-I or II. In the presence of PC, however, both enzymes caused 100% hemolysis even at as low as 0.1 mg. These results suggested that both enzymes are negative or very weak as to direct hemolytic activity, while they exhibit indirect hemolytic activity through the lyso-PC produced from the cleavage at the sn-1 or 2 ester bond of PC. Furthermore, TLC analyses demonstrated that the reaction of each puri®ed enzyme with the ¯uorescent PC labeled at the sn-2 position resulted in the liberation of ¯uorescent fatty acids. Thus, the two puri®ed enzymes were con®rmed to be PLA2s speci®cally hydrolysing the sn-2 ester bond of phospholipids. The optimum pH values of AP-PLA2-I and II were estimated to be 9.0 and 8.0, respectively. The activity of both enzymes was enhanced to about 180% by Ca2+ but reduced to 10±20% by Cu2+ and Zn2+, as compared to that determined without divalent cations. Neither enhancement nor reduction of the enzyme activity was displayed by Fe2+, Mg2+ and Mn2+. The molecular masses of native AP-PLA2-I and II were determined to be 28 and 12 kDa, respectively, by gel ®ltration HPLC. On the other hand, both PLA2s gave a single band corresponding to a molecular mass of 15 kDa on SDS-PAGE in the presence of a reducing agent (Fig. 4). These results indicated that AP-PLA2-I is a dimer composed of the same subunit, while AP-PLA2-II is a monomer. As shown in Table 2, the amino acid compositions of the two enzymes were very similar to each other; Asx, Glx and Gly were predominant in both molecules but Met, His and Trp were poor. Analyses by a sequencer identi®ed the ®rst 62 amino acid residues for both enzymes (Fig. 5). A marked homology (65%) was seen between the N-terminal sequences of the two enzymes; as many as 40 of the 62 residues were identical and all the 7 Cys residues were conservatively located. No impurities were detected in each cycle of the sequencing of AP-PLA2-II. In the AP-PLA2-I preparation, however, minor amino acids were ad-

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Fig. 4. SDS-PAGE of the puri®ed PLA2s. SDS-PAGE was performed in the presence of 2-mercaptoethanol. Lane A, standard proteins; lane B, AP-PLA2-I; lane C, AP-PLA2-II.

ditionally found at 17 positions, suggesting the coexistence of at least one minor PLA2 component. Although most of the minor amino acid residues detected at 17 positions were the same as the residues at the corresponding positions of AP-PLA2-II, there were apparent di€erences at positions 40 and 58 between the minor PLA2 component and AP-PLA2-II. Therefore, the minor PLA2 component contained in the AP-PLA2-I preparation was judged to be not due to the contamination of AP-PLA2-II.

DISCUSSION

In this study two PLA2s (AP-PLA2-I and II) were puri®ed from the A. planci venom and their properties were clari®ed. A minor PLA2 component was additionally found in the AP-PLA2-I preparation. Succeeding to the lethal factor (Shiomi et al., 1988) and the anticoagulant factor (Karasudani et al., 1996), these PLA2s are the third class of biologically active substances puri®ed from the A. planci venom. Table 2. Amino acid compositions of AP-PLA2-I and II Mol% Amino acid Asx Thr Ser Glx Pro Gly Ala Val Cys (1/2) Met Ile Leu Tyr Phe His Trp Lys Arg

AP-PLA2-I

AP-PLA2-II

9.9 7.5 6.2 10.6 3.1 11.6 5.6 4.4 7.7 0.5 3.6 5.1 6.7 3.6 0.9 1.5 7.4 4.2

11.9 6.1 4.0 10.3 2.4 12.7 7.0 5.8 8.3 0.8 2.6 4.9 7.5 3.3 1.0 0.7 6.0 4.7

Fig. 5. Comparison of the N-terminal amino acid sequences of the A. planci PLA2s with those of some classes I and II enzymes. The minor amino acids detected in the AP-PLA2-I preparation are shown below the sequence of AP-PLA2-I. Class I enzymes: notexin from Australian tiger snake (Halpert and Eaker, 1975), Taiwan cobra PLA2 (Tsai et al., 1981) and bovine pancreatic PLA2 (Fleer et al., 1978). Class II enzymes: himehabu PLA2 (Joubert and Haylett, 1981) and mamushi PLA2 (Forst et al., 1986). The residues which are identical with AP-PLA2-I are boxed and those which are identical only with AP-PLA2-II are shaded.

596 K. SHIOMI et al.

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597

AP-PLA2-I and II are comparable to each other in many points. Their enzyme activities are highest on a weakly alkaline side (pH 8 or 9) and are enhanced in the presence of Ca2+. They are neutral or acidic proteins. There is a high homology (65%) between their N-terminal sequences (up to the 62nd residue). However, they are separable from each other by hydrophobic chromatography on Phenyl Sepharose CL-4B; the behaviors on Phenyl Sepharose CL-4B suggest that AP-PLA2-I is lower in hydrophobicity than AP-PLA2-II. They also di€er from each other as to the subunit structure. AP-PLA2-I is a dimeric protein composed of the same subunit, while AP-PLA2-II is a monomer. Based on their primary structure and disul®de bonding pattern, extracellular PLA2s from various sources are divided into three classes as follows: class I enzymes from elapid and hydrophid snake venoms and mammalian pancreas, class II enzymes from crotalid and viperid snake venoms and class III enzymes from lizard and bee venoms (Arni and Ward, 1996). The determined N-terminal sequences reveal that both AP-PLA2-I and II are new members of class I enzymes, being distinct from classes II and III enzymes. Since classes I and II enzymes are fairly homologous with each other, the Nterminal sequences of three class I enzymes, notexin from Australian tiger snake (Halpert and Eaker, 1975), Taiwan cobra PLA2 (Tsai et al., 1981) and bovine pancreatic PLA2 (Fleer et al., 1978), and two class II enzymes, himehabu PLA2 (Joubert and Haylett, 1981) and mamushi PLA2 (Forst et al., 1986), are aligned with those of the A. planci PLA2s in Fig. 5, in order to make it easy to understand that the A. planci PLA2s are class I enzymes. Class I enzymes are clearly distinguishable from class II enzymes with respect to the location of Cys residues; Cys-11 is characteristic of only class I enzymes and Cys-51 of only class II enzymes (Arni and Ward, 1996; Heinrikson, 1991). Obviously, both AP-PLA2-I and II possess Cys-11 but lack Cys-51. Another prominent di€erence between classes I and II enzymes resides in the region 53±61 put between the two Cys residues (Cys-52 and Cys-62); class II enzymes have 6 amino acids in this region, while class I enzymes display an insertion of two or three amino acids (so-called `elapid loop') (Arni and Ward, 1996; Heinrikson, 1991). In accordance with class I enzymes, both AP-PLA2-I and II appear to have the elapid loop. The fact that the A. planci PLA2s contain Thr-37, which is commonly found in only class I enzymes, further supports that they are class I enzymes. It is helpful to compare in more detail the N-terminal sequences of the A. planci PLA2s with those of the known classes I and II PLA2s. The 22 residues (Leu-2, Gln-4, Phe-5, Tyr-26, Gly-27, Cys-28, Cys-30, Gly-31, Gly-33, Gly-34, Gly-36, Pro-38, Asp-40, Asp-43, Arg-44, Cys-45, Cys-46, His-49, Asp-50, Cys-52, Tyr-53 and Cys-62), which are known to be absolutely or highly conserved for both classes I and II PLA2s (Heinrikson, 1991), are certainly observed in the A. planci PLA2s. Accordingly, the statement of Arni and Ward (1996) that the sequences of the catalytic site (positions 45±52) and the calcium binding loop (positions 29±33) are CCxxHDxC and W/ YCGxG, respectively, in common with classes I and II enzymes, is true for both APPLA2-I and II, except that AP-PLA2-II possess Phe at position 29. However, the A. planci PLA2s are unique in that they have an insertion of one amino acid at an unknown position between Cys-11 and Tyr-26, di€ering from the known class I enzymes. It should be also pointed out that the Val residue at position 9 in both APPLA2-I and II and the Phe residue at position 29 in AP-PLA2-II are rather unusual. Ile is generally located at position 9 and Tyr at position 29 in the known classes I and II PLA2s (Heinrikson, 1991). In the case of three PLA2s from the green habu snake Trimeresurus gramineus, the replacement of Tyr by Phe at position 29, which constitutes the calcium binding loop, has been reported to have no e€ect on the binding of Ca2+

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(Oda et al., 1991; Fukagawa et al., 1993). This seems to be the case with AP-PLA2-II, since its enzyme activity is rather high compared to AP-PLA2-I with Tyr-29. Whether the A. planci PLA2s have biological activities as well as enzyme activity, similar to snake venom PLA2s, is of particular interest. In this relation, we have preliminarily found that both AP-PLA2-I and II exhibited capillary-permeability-increasing activity when injected into the rat back skin at a dose of 10 mg. Since the capillarypermeability-increasing activity has previously been shown to be signi®cantly reduced by H-1 antihistaminics and in addition suggested to be due to histamine released from mast cells by histamine-releasing factors in the A. planci venom (Shiomi et al., 1989), it is very likely that the histamine-releasing factors are none other than the PLA2s under study. On the other hand, both AP-PLA2-I and II caused no edema in the mouse foot pad even at a dose of 20 mg, indicating the occurrence of unknown edema-forming factors in the A. planci venom. In fact, it has been reported that the edema-forming activity displayed by the crude toxin is not inhibited by any antihistaminics (Shiomi et al., 1989). To our knowledge, the PLA2s so far puri®ed from marine venoms are limited to those from the snail Conus magus (McIntosh et al., 1995) and the jelly®sh Rhophilema nomadica (Lotan et al., 1995). Unlike the A. planci PLA2s, these enzymes are homologous with lizard PLA2s (class III enzymes). In conclusion, the A. planci PLA2s are the ®rst class I enzymes of marine venom origin. Studies on their detailed biological activities and complete amino acid sequences are now in progress. Acknowledgements ÐThe authors thank Dr A. Shinagawa, Gakushuin Women's College, for measuring the amino acid compositions and Mr M. Fukushima and Mr K. Ikeda in our laboratory for their technical assistance.

REFERENCES Arni, R. K. and Ward, J. (1996) Phospholipase A2: a structural review. Toxicon 34, 827±841. Bhat, M. K. and Gowda, T. V. (1989) Puri®cation and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom. Toxicon 27, 861±873. Fleer, E. A. M., Verheij, H. M. and de Haas, G. H. (1978) The primary structure of bovine pancreatic phospholipase A2. Eur. J. Biochem. 82, 261±269. Forst, S., Weiss, J., Blackburn, P., Frangione, B., Goni, F. and Elsbach, P. (1986) Amino acid sequence of a basic Agkistrodon halys blomhoi phospholipase A2. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli. Biochemistry 25, 4309±4314. Fukagawa, T., Nose, T., Shimohigashi, Y., Ogawa, T., Oda, N., Nakashima, K., Chang, C.-C. and Ohno, M. (1993) Puri®cation, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon 31, 957±967. Halpert, J. and Eaker, D. (1975) Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake). J. Biol. Chem. 250, 6990±6997. Heinrikson, R. L. (1991) Dissection and sequence analysis of phospholipases A2. Methods Enzymol. 197, 201± 214. Joubert, F. J. and Haylett, T. (1981) Puri®cation, some properties and amino acid sequence of a phospholipase A2 (DE-I) from Trimeresurus okinavensis (hime-habu) venom. Hoppe-Seyler's Z. Physiol. Chem. 362, 997± 1006. Karasudani, I., Koyama, T., Nakandakari, S. and Aniya, Y. (1996) Puri®cation of anticoagulant factor from the spine venom of the crown-of-thorns star®sh Acanthaster planci. Toxicon 34, 871±879. Lotan, A., Fishman, L., Loya, Y. and Zlotkin, E. (1995) Delivery of a nematocyst toxin. Nature 375, 456. Lowry, L., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265±275. Marinetti, G. V. (1965) The action of phospholipase A on lipoproteins. Biochim. Biophys. Acta 98, 554±565. McIntosh, J. M., Ghomashchi, F., Gelb, M. H., Dooley, D. J., Stoehr, S. J., Giordani, A. B., Naisbitt, S. R. and Olivera, B. M. (1995) Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus. J. Biol. Chem. 270, 3518±3526.

Phospholipase A2 of A. planci

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Oda, N., Nakamura, H., Sakamoto, S., Liu, S.-Y., Kihara, H., Chang, C.-C. and Ohno, M. (1991) Amino acid sequence of a phospholipase A2 from the venom of Trimeresurus gramineus (green habu snake). Toxicon 29, 157±166. Shiomi, K., Itoh, K., Yamanaka, H. and Kikuchi, T. (1985) Biological activity of crude venom from the crown-of-thorns star®sh Acanthaster planci. Nippon Suisan Gakkaishi 51, 1151±1154. Shiomi, K., Yamamoto, S., Yamanaka, H. and Kikuchi, T. (1988) Puri®cation and characterization of a lethal factor in venom from the crown-of-thorns star®sh (Acanthaster planci). Toxicon 26, 1077±1083. Shiomi, K., Nagai, K., Yamanaka, H. and Kikuchi, T. (1989) Inhibitory e€ect of anti-in¯ammatory agents on cutaneous capillary leakage induced by six marine venoms. Nippon Suisan Gakkaishi 55, 131±134. Shiomi, K., Yamamoto, S., Yamanaka, H., Kikuchi, T. and Konno, K. (1990) Liver damage by the crown-ofthorns star®sh (Acanthaster planci) lethal factor. Toxicon 28, 469±475. Shiomi, K., Saitsu, N. and Kikuchi, T. (1991) Hepatotoxic e€ects induced in rats by the crown-of-thorns star®sh Acanthaster planci lethal factor. Nippon Suisan Gakkaishi 57, 341±344. Shiroma, N., Noguchi, K., Matsuzaki, T., Ojiri, Y., Hirayama, K. and Sakanashi, M. (1994) Haemodynamic and haematologic e€ects of Acanthaster planci venom in dogs. Toxicon 32, 1217±1225. Taira, E., Tanahara, N. and Funatsu, M. (1975) Studies on the toxin in the spines of star®sh Acanthaster planci. I. Isolation and some properties of the toxin found in spines. Ryukyu Daigaku Nogakubu Gakujutsu Hokoku 22, 203±213. Tsai, I.-H., Wu, S.-H. and Lo, T.-B. (1981) Complete amino acid sequence of phospholipase A2 from the venom of Naja naja atra (Taiwan cobra). Toxicon 19, 141±152. Vishwanath, B. S., Kini, R. M. and Gowda, T. V. (1987) Characterization of three edema-inducing phospholipase A2 enzymes from habu (Trimeresurus ¯avoviridis) venom and their interaction with the alkaloid aristolochic acid. Toxicon 25, 501±515. Yara, A., Noguchi, K., Nakasone, J., Kinjo, N., Hirayama, K. and Sakanashi, M. (1992) Cardiovascular e€ects of Acanthaster planci venom in the rat: possible involvement of PAF in its hypotensive e€ect. Toxicon 30, 1281±1289.