Isolation of proteinase inhibitory, toxic and hemolytic polypeptides from a sea anemone, Stoichactis sp.

0041-0101/80/0101-0097502.00/0

Tadcaw, VoL 18, pp . 97-106. ~ ParBamon Prat Ltd 1980. Prlnttd is Great Britain

ISOLATION OF PROT'EINASE INHIBITORY, TOXIC AND HEMOLYTIC POLYPEPTIDES FROM A SEA ANEMONE, STOICHACTIS SP. D. MESS

and E. GEBAUER

Zentrum der Rechtsmedizin, University of Frankfurt, Frankfurt/Main, dermany (dcceptedfor publication 12 June 1979) D. M>:es and E. (3sBnrreß. Isolation of proteinase inhibitory, toxic and hemolytic polypeptides from a sea anemone, Stoichactis sp . Toxicon 18, 97-106, 1980 .-From a sea anemone, Stoichactis sp ., biologically active polypeptides exhibiting toxic, hemolytic and proteinase inhibitory properties have been isolated by gel filtration and ion-exchange chromatography. For the hemolytic polypeptide a mol. wt of 10,000 was determined by gel filtration . It indtwes complete hemolytis of human erythrocytes at 0"86 ltg/ml wncentration, is free of phospholipase A activity and has ichthyotoxic activity . The toxic principle (minimum lethal dose ca. 2"3 mg/kg injected s.c. into mice, and 0"5 mg/kg for crabs, i.m. injection) has a mol. wt of about 6000 and was separated from the hemolytin and from the three proteinase inhibitors which inhibit trypsin and chymotrypsin and which are composed of 52-56 amino acid residues . INTRODUCTION

sea anemone nematocysts, tentacles or even the whole animal, various biologically active factors have been isolated . Neuro- and cardiotoxic polypeptides were purified from Anemonia sincere (BERESS et al., 1975), Condylactis aurantiaca (BERESS et al., 1976), and Anthopleura xanthogrammica and A . elegantissima (SHIBATA et al., 1976 ; NORTON et al., 1978), hemolytic factors from Aiptasia pallida (HESSINQER and LExi-ZOFF, 1973) and proteinase inhibitors from Anemonia sincere (FRITZ et al., 1972). Sea anemones of the genus Stoichactis are large specimens and are quite abundant on the coral reefs of the Indo-Pacific region and adjacent seas . They are well known by their symbiotic relationships with certain reef fishes (Amphiprion, family Pomacentridae) . TURLAPATY et al. (1973) described the isolation of a central stimulant substance from Stoichactis kenti. A toxin which was partially purified from Stoichactis helianthus possessed, beside lethal activity (LD3o 0"25 mg/kg mouse, i.p. injection), hemolytic properties (DEVLIIV, 1974) which were specifically inhibited by sphingomyelin (BERNHEIh1ER and AVIGAD, 1976). In the present study the isolation and characterization of toxic, hemolytic and proteinase inhibitory principles from a Stoichactis species are described. FROM

MATERIALS AND METHODS Sea anemones of the genus Stoichactis from the Indo-Malayan coast were obtained from a tropical fish importing company in the frozen state and stored at -20°C until use. Due to the oanditions of the animals after thawing exact taxonomic specification could not be performed. Pur~rntion andfractionationprocedures In a typical experiment sea anemones (about 1 kg) were extracted 4 times with distilled water (3 1) with slow stirring after thawing. The combined extracts were filtered through a layer of glass wool to remove macerated tissues. To the filtrate, 2"S volumes of cold acetone (-10°C) were added. The solution was kept 97

98

D. MEBS and E. GEBAUER

at 4°C for 12 hr. The precipitate formed was collected by centrifugation at 6000 rev/min for 20 min at 0°C and washed twice with cold acetone. The clear, yellow supernatant was discarded and the precipitate dissolved in O~1M ammonium acetate buffer, pH 6~8 ; the insoluble part was removed by centrifugation . The brown supernatant was applied onto a Sephadex G-75 column (110 x 5 cm) which had been equilibrated with 0~1 M ammonium acetate buffer, pH 6~8, andeluted using the same buffer. Two active fractions showing hemolytic and toxic as well as proteinase inhibiting properties were obtained and lyophilized. The hemolysin was further purified by chromatography on a SP-Sephadex C-25 column (3~5 x 1~0 cm) using a linear gradient of 005 M ammonium acetate buffer pH 5~3 to 0~2 M, pH 6~8 or by stepwise elution (0~1, 0~2 and 0~5 M, pH 6~8). Toxic and proteinase inhibitory activities were separated by chromatography of the freezedried Sephadex-fraction on CM~ellulose using a similar gradient (005 M ammonium acetate buffer, pH 5~5, to 0~5 M, pH 6~8) . The three proteinase inhibitors isolated were further purified by chromatography on SP-Sephadex C-25 (identical elution gradient). Hemolytic activity

Human erythrocytes washed three times with physiological saline were used in 0~6 % suspension . Hemolytis was tested at 25°C by addition of various concentrations of hemolytic fractions to 5 ml erythrocyte suspension. The concentration of the hemolysin necessary to produce wmplete hemolytis within 10 min was determined. Phospholipase A activity

This was assayed according to the method Of MARINETTI (1965) using the clearing of an egg yolk solution .

Proteinase inhibition

Inhibition of trypsin (TPCK-treated trypsin, Merck, Darmstadt) was tested using a-N-toluensulfonyl-Larginine methylester (TAME) and of chymotrypsin (Boehringer, Mannheim) using a-N-benzoyl-t-tyrosine ethylester (BTEE) as substrate. Enzyme solution in 005 M Tris-HCl buffer (0~1 ml) pH 8~0 was incubated with various inhibitor concentrations for 20 min at 25°C and the residual proteinase activity determined spectrophotometrically (increase of absorbency at 247 nm for TAME and at 256 nm for BTEE). One inhibitory unit was defined as the amount of protein to inhibit one unit of enzyme activity. Lethality tests

Lethality of fractions and purified material was tested by s.c . injection into mice (20 g) and by i.m . injection into shore crabs (Carcinus mamas, 10 g) . For the investigation of ichthyotoxic activity, small Killifishes (Poecilia reticulate, l cm)were submerged in tap waterwhere the toxic material had been dissolved. Protein concentration

This was determined according to the method of

LOWRY et al.

(1951) .

Disc electrophoresis

This was carried out in polyacrylamide gel (7~5 ~) at pH 4~3 for 90 min at 8 V per tube ; the gels were stained with Amino black IOB. Molecular weight determination

The estimation of the mol. wt was performed by gel filtration on a calibrated Sephadex G-75 wlumn with 0~ 1 M Tris-HCl buffer pH 8~0 using a-bungarotoxin (own preparation), cytochrome c, a-chymotrypsinogen and ovalbumin (Serve, Heidelberg) as standard compounds. Determination of amino acid composition and N-terminus

Protein samples were hydrolyzed for 24 hr at 110°C in vacuum-sealed tubes with constant boiling glassdistilled 5~7 N HCI. Amino acid analysis was kindly performed by Dr . Y. Samejima, Pharmacological Institute, University of Giessen using a Biotronik amino acid analyzer LC 6000 (Franlâurt, Germany) . Tryptophan was determined after 24 hr methanesulfonic acid hydrolysis according to the method of Su+esox et al. (1976) . The amino terminal residue of the native proteinase inhibitors was determined by Edman-degradation according to the method of IwnNaan et al. (1969) . RESULTS

Isolation ofbiologically active polypeptides Extraction of Stoichactis sea anemones after thawing with distilled water (producing

osmotic cell disruption) proved to be more useful (higher yields, less inactive material) than homogenisation of the whole animal in 30 ~ alcohol or distilled water. After precipitation of the protein from the extract with cold acetone, the supernatant was free of hemolytic, toxic and proteinase inhibitory properties . By gel filtration of the dissolved precipitate on Sephadex G-75, two active fractions were separated : one possessing hemolytic activity and another, sometimes overlapping, which

Polypeptides from a Sea Anemone

99

produced death in mice and crabs and inhibited the proteolytic activity of trypsin and chymotrypsin . Figure 1 shows the elution pattern of a small column . Using larger columns for preparative purpose (110 x 5 cm) the two combined fractions can be better separated (hemolytic from toxic and proteinase inhibitory activity) by repeating the gel filtration on a second column after lyophilisation. The hemolysin was further purified by chromatography on SP-Sephadex C-25 (Fig. 2). It is not clear if there is one substance or two, since, in disc electrophoresis at pH 4~3, both hemolytic fractions behaved similarly (Fig. 3). aoo E

Io

_o 200

Hemdysin

a

L C

Tube No .

Inhibitor + toxin

FIG. l . GEL FILTRATION OF ACETONE PRECIPTCATE FROM StOIChQCtIS SP. EXTRACT ON A SEPHADEX G-75 coLUMN usnvG 0~1 M AMMONIUM wcerATE, pH 6"8.

Hemolytic activity was tested on washed human erythrocytes ; proteinase inhibitory properties (dashed line) on trypsin and toxic activity by s .c . igjection into mice . Fractions of 6 ml were collected at a flow rate of 40 ml/hr.

10

20

30 Tube

40 No.

SO

FIG . T. PURIFICATION OF THE HHMOLYSIN HY CHROMATOGRAPHY ON A SEPHADEX GZS COLUMN usuaG w Ln~rEAR GRADZeNT of 0"OS M wMMOxnIM ACETATE HuFFER, pH 5~3 To 0"2 M, pH 6"8, AND STEPWISE ELUTION HY O'S M HUFFER .

By chromatography on CM-cellulose or SP-Sephadex C-25 the toxin was separated from the proteinase inhibiting activity which was resolved into three inhibitors by various steps of rechromatography (Fig. 4). Because of the very low concentration of the toxin, further purification was not performed. Froteinase inhibitors 2 and 3 were homogeneous in disc electrophoresis while inhibitor 1 and the toxic fraction were impure (Fig. 3). Properties of the hemolysin

The mol. wt of the hemolysin was determined by gel filtration on a calibrated Sephadex G-75 column to be about 10,000 (Fig. 5). The protein did not show phospholipase A activity nor was lecithin or serum required to produce hemolysis. Therefore, the sea anemone factor can be classified as a direct hemolysin. Complete hemolysis of a 0~6 ~ human erythrocyte suspension was achieved at a concentration of 086 ~g/ml. When added to small fish

100

D . MEBS and E. GENAUER O'O5M, pH5'5-0'2M, pH 6 8 ammonium acetate

n0 0'8 0 mN

a

0'6

o'a o'z

F-0'05M, pH5'B-O'SM, pH6 8 ammonium acetate 0'2 0' I

0' 4

00 N a

0'2

0'2

zo

ao

Tube

so

No .

eo

FIß . 4. SEPARATION OF THE TOXiC AND PROTEINASE INHIHTTORY PRINCIPL88 FRACTION OF ßEL FILTRATION BXPERiMENT, >~iß . 1) HY CHROMATOßRAPHY ON A CM~,LULOSE COLUMN (9'S X l'0 cm, a) AND PURIFICATION OF THREE PROTEINASE INHIBITORS (b, c, d) oN SP-~!~ "nEx G25 COLUMNS (S X I ~), USR~Iß AMMONIUM ACETATE HUPFER ßRADDiNTS POR ELUTION.

Solid line-absorbency at 280 nm, dashed line-proteinase inhibitory activity.

tanks, it is able to kill fishes (1 cm in size), in a concentration of ~S lIg/ml, within 1 hr. On the other hand, mice survive i .p . injections of 200 ~g without pathological symptoms . Properties of the toxic principle Since lethal activity eluted in the same fraction as the proteinase inhibitors, a mol . wt of about 6000 is suggested . Further chemical characterization could not be performed at this stage due to the very small amount of material available which proved to be impure in disc electrophoresis (Fig . 3) . However, for this fraction a minimum lethal dose of about 2~3 mg/kg was determined for mice by s .c . injection . The animals first screamed and jumped

I

Inhibitor 2

Toxin

3

Hemolysin

FIG. 3 . DISC ELECTROPHORESIS OF PROTEINASE INHIBITORY, TOXIC AND HEMOLYTIC POLYPEPTIDES FROM StOIChQCtIS 3P., ~ GEL, pH 4'3, ô V PER TUBE FOR 90 min.

i's

Polypeptides from a Sea Anemone

2~2

~~

103

Inhiblior 2 (mol . wr 5800)

2~ 0

1~4 3'8

40

42

Log. moh wi

4 "i

4~6

FIO . S. MOIBCUIAR wEIOHT D6TSRI~IINATION OF TfR; HCMOLYSIN AND Tfn3 PR01'EIIdA38 INFIIBTrOR (nvmerroR 2) BY OEL PII.TRATION A CALIHRATED SePxADac G-75 wLUMN (135 x 1 "5 cm) usnvo a-HVNaARO~roxnv (8000 mol. wt), cnnctmoME c (11,700), a-cx~orRrPSnvoorav (25,700) AND ovALSV~r (43,000) As srANDARD co~ouNDS, V, = ELVTCON voLUa~, V, = vom voLUt~ .

probably due to local pain and soon developed paralysis of the hind legs ; death appeared due to respiratory arrest . The minimum lethal dose for shore crabs (Carcinus muenas) was lower, about 0"5 mg/kg; the toxic material was injected i.m . into the walking legs . Within minutes the crabs showed into-ordinated movement and convulsive reactions of the extremities . The toxin has no ichthyotoxic properties when tested in concentrations up to 50 ~g/ml on Killi fishes . Properties of the proteinase inhibitors Table 1 shows the amino acid composition of inhibitors 2 and 3 calculated on a basis of about 6000 mol. wt . For inhibitor 2, the main component, a mol. wt of 5800 had been determined by gel filtration (Fig . 5) . All polypeptides lack tryptophan and contain 4 (inhibitor 2) and 6 (inhibitor 3) half~ystinyl residues. By Edman-degradation no N-terminal amino acid reactive to phenylisothiocyanate could be detected for the two inhibitors suggesting a masked N-terminus . By incubation of enzyme (trypsin and chymotrypsin) and inhibitor for 20 min, optimal inhibition was achieved ; longer incubation times did not increase the inhibitory rate . Whereas inhibitors 2 and 3 inactivated trypsin almost completely (98 ~)andtoalesserextent chymotrypsin (86 and 92 ~, respectively), inhibitor 1 exhibited relatively low activity (80 ~ inhibition of trypsin, 50 ~ of chymotrypsin). If the molar ratio of enzyme : inhibitor is calculated on the basis of inhibitory titration curves, it is confirmed that inhibitor 3 is the most active, exhibiting a ratio of 1 :1 for trypsin and 1 :1 "2 for chymotrypsin . Inhibitor 2 shows a ratio of 1 :1 "4 and 1 :1 "8; inhibitor 1 a ratio of 1 :1 "8 and 1 :1, but in the latter case chymotrypsin is only inhibited 50 ~. DISCUSSION

Bioactive substances from marine organisms attract growing interest especially because of their potential pharmaceutical value (cf. KAUL and SINDERbIANN, 1978). Exploration of the

10 4

D. MEBS and E. GEBAUER TABLE I . AMIIVO ACID COMPOSITION OF TWO PROTEIIVASE INHIBTIC)RS ISOLATED FROM S10ÎClICICtIS SP. SAMPLES WERE FIYDROLYZED FOR 24 hi AT 11O°C n~r 5~7 N HCI. TRYPTOPeAN WAS DETERMINED AFTER 24 hr METHANE.4ULFONIC ACID HYDROLYSIS

Amino acids Asp Thr Ser Glu Pro Gly Ala } Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp Total Formula weight

Residues per mole Inhibitor 2 Inhibitor 3 4~0 (4) 4~7 (~ 2~4 (3) 2~5 (3) 1~7 (2) 1~5 (2) 3~5 (4) 3ß (3) 3~0 (3) 0~5 (1) 6~8 (~ 8~3 (8) 3~1 (3) 3~3 (3) 4~1 (4) 5~4 (6) 1~6 (2) 1~7 (2) 0 0~5 (1) 3~1 (3) 3~6 (4) 2~5 (3) 2~1 (2) 2~1 (2) 2~1 (2) 3~3 (3) 3~3 (3) 3~7 (4) 3~9 (4) 0~9 (1) 1~2 (1) 4~2 (4) 62 (6) 0 0 52 56 5690 6188

sea for new natural products having antimicrobial, cytotoxic and other pharmacological properties is still in its beginnings. Of the sea anemones only a few species have been thoroughly investigated . It is not surprising that a number of highly active substances have been isolated, since these animals, belonging to the Cnidaria, possess very specialized cells (nematocysts) which are used for prey capture. Fishes or small invertebrates are paralyzed or at least immobilized upon discharge of the nematocysts. Methods for fractionation of sea anemone extracts and separation of the active components of protein nature are well established ($HAP1R0, 1968 ; BERESS and BERESS, 1971 ; BSRESS et al., 1975, 1976 ; CARIELLO arid D'ANISLLO, 1975). By these procedures we were able to isolate three distinct principles from the sea anemone 5toichactis sp. : a toxin, hemolysin (s) and proteinase inhibitors . In contrast to previous investigations on Stoichactis helianthus where hemolytic and lethal activities were identical (DBVLIN, 1974; BeRxxetMeR and AVIGAD, 1976) and associated with a protein of 16,000 mol. wt (similar observations were made by FERLAN and LEBEZ, 1974, on equinatoxin from Actinic eguina), both activities could be completely separated. The hemolysin is a basic polypeptide of about 10,000 mol. wt and lacks any lethal activity towards mice, whereas the toxin or the toxins (at the present state of purification the existence of additional active components cannot be excluded) shows a mol. wt between 5000-6000, resembling in this respect toxins from Anemonia sulcata (BERess et al., 1975) and from Condylactis aurantiaca (BERESS et al., 1976). This toxic factor displays also higher lethal activity (about 5 times) towards crustaceans than to mice. For Anemonia sulcata toxins a specific neurotoxic action in crustaceans has been reported (RATHHIAYeR et al., 1975), but in mammals pronounced cardiotoxic actions predominate (ALSEN et al., 1976, 1978). Whether this is also the case for Stoichactis toxin, can only be answered when more pure material becomes available.

Polypepddes from a Sea Anemone

lOS

The sea anemone's hemolytic factor is free of phospholipase A activity, since neither lecithin nor serum is required to produce complete hemolysis of human erythrocytes . Regarding its hemolytic concentration (086 ~g/ml), it is less active than the factor from Stoichactis helianthus (0~1 ltg/ml, BSRNHEIMER and Avia.,D, 1976), but has higher activity than classical agents like lysolecithin (4 4tg/ml, Hn$aRa~tnxrr, 1957) or melittin from bee venom (4 ~g/ml, HASaRMaxx and KOWALLEK, 1970). Whether it has an affinity to specific constituents of the red cell membrane, such as to sphingomyelin like the Stoichactis helianthus factor, or whether it produces cell lysis by directly disordering the lipid bilayer in a `detergent'-like manner as melittin does, has to be investigated. The ichthyotoxic properties of the hemolysin seem to be related to and involved in the cell lytic action . PRIMOR and ZL.OTäIN (197 isolated a similar polypeptide having hemolytic as well as ichthyotoxic activity from the skin secretion ofthe flatfish Pardachirus marmoratus . Moreover, melittin is also a potent ichthyotoxin (PRIMOR and ZLOTKIN, 197 . This leads to the assumption that these hemolytic factors may affect any membrane in the fish, such as the gill membrane which is the most sensitive organ due to its direct exposure. Toxin and hemolysin when discharged by the nematocysts capturing mechanism may serve as powerful agents for prey acquisition. On the other hand, the presence of proteinase inhibitors in the tissue of the sea anemone is difficult to explain. At least three were isolated from Stoichactis sp. (some minor components have not been further characterized). FxiT2 et al. (1972) and Wuxn~ReR et al. (1976) described the isolation of 10 inhibitors from Anemonia sulcata . All these polypeptides have strong inhibitory effects on trypsin, chymotrypsin, plasmin and kallikrein and belong to the group of basic trypsin-inhibitors originally isolated from bovine organs (Bl'T'I or Kunitz-inhibitor, KASSELL and LASxowsxi, 1965). A number of similar polypeptides have been purified from various sources (snails, spermatozoans, snake venoms, cf. FRiTZ et aL, 1974) having in common 6 half-cystinyl residues and lacking tryptophan . The amino acid composition of inhibitor 2 from Stoichactis sp. shows close similarities to an inhibitor (Ia) from the venom of the snake Naja nivea (HoxnMn et al., 1976). Since both have only 4 half-cystinyl residues they may differ in conformation from those with 6 residues. It is also interesting to note that the Stoichactis inhibitors show increasing inhibitory capacity with increasing basic properties. Sequence studies on inhibitor 3 are in progress. The biological significance of proteinase inhibitors in sea anemones is unclear. They may protect the animal from digestion by proteases of the prey or, since they are supposed to be applied together with the hemolysin and the toxic principle, they may protect these factors from proteolytic inactivation by the prey . It is also possible that the inhibitors play a role in the regulation of the animals digestive mechanisms preventing for instance selfdigestion by its own proteolytic enzymes or by those of symbiotic zoochlorellas. However, these are speculations, since our knowledge of coelenterate physiology, especially their digestive metabolism is rather fragmentary (cf. MUSCATINB and Lsxxor>?, 1974). Although exact taxonomic specification of the sea anemone cannot be given in the present study due to the bad condition of the thawed material, the results should encourage further investigations on this common species (Stoichactis, Radianthus) widely distributed along tropical coastlines. Acknowledgements-The authors are indebted to Mr. WICxER for providing the sea anemones . T'ho present work has been partially supported by the Edinger-Foundation.

REFERENCES Arw, C., B8RFS3, 1. ., FrscHER, 1C ., PROFPF, D ., REQVHERO, T, and $A11LER, R . W. (197 The action Of S toxin from the sea anemone Anemonia sulcata upon mammalian heart muscles . Naunyn-Schmtedeberg â Arch . exp. Path. Pharmac. 295, SS .

10 6

D. MEBS and E. GEBAUER

Arsvar, C., B~eESS, L. and Tessettwux, I. (1978) Toxicities of sea anemone (Anemonia sulcata) polypeptides in mammals. Toxicon 16, 561. B~eFSS, L. and Battrss, R. (1971) Reinigung zweier Krabben lähmender Toxine sus der Seeanemone Anemonia sulcata. Kieler Meeresforsch . 27, 117. BexFSS, L., Bsts~ss, R, and WuxnExEx, G. (1975) Purification of three polypeptides with neuro- and cardiotoxic activity from the sea anemone Anemonia sulcata. Toxicon 13, 359. BERFSS, R., BSRFS3, L. and Wut~t.xm, G. (1976) Neurotoxins from sea anemones . Purification and characterization of four polypeptides with neurotoxic activity from Condylactis aurantiaca. HoppeSeyler's Z. physiol. Chem . 357, 409. BERNHEIIdER, A. W. and Aviawv, L. S. (1976) Properties of a toxin from the sea anemone Stolchactis helianthus, including specific binding to sphingomyelin. Proc. natn. Acad. Sci. U.S.A . 73, 467. Cwx~u..o, L. and D'Ai~i t.o, A. (1975) Isolation and characterization of four toxic protein fractions from the sea anemone Anemonia sulcata. Toxicon 13, 353. Dsvtnv, J. P. (1974) Isolation and partial purification of hemolytic toxin from sea anemone, Stoichactls helianthus. J. Pharm. Sci. 63, 1478 . Fexr.wrr, I. and LESez, D. (1974) Equinatoxin, a lethal protein from Actinic equina-I . Purification and characterization. Toxicon 12, 57 . Fnrrz, H., BREY, B. and B6RFS4, L. (1972) Polyvalente Isoinhibitoren fir Trypsin, Chymotrypsin, Plasmin and Kallikreine aus Seeanemonen, (Anemonia sulcata), Isolierung, Hemmverhalten and Aminosäure zusammensetzung. Hoppe-Seyler's Z. physiol. Chem . 353, 19. Fxrrz, H., Tscxescxe, H., GR~, L. J. and Txusctu:rr, E. (Eds . 1974) Proteinase Inhibitors . Proc. 2nd Int. Res. Conf., Bayer Symp. V, Springer, Heidelberg. HwHEantwivN, E. (1957) Beiträge zur Pharmakologie von Phospholipase A. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 230, 538. HABERMANN, E. and Kowwc.tetc, H. (1970) Modifikation der Aminogruppen and des Tryptophans im Melittin als Mittel zur Erkennung von Struktur-Wirkungs-Beziehungen . Hoppe-Seyler's Z. physiol. Chem . 351, 884. HessuaGEx, D. A, and LExHO~, H. M. (1973) Assay and properties of the hemolytic activity of pure venom from the nematocysts of the acontia of the sea anemone Aiptasia pallida. ArchsBiochem. Biophys.159, 629. HoxwMw, Y., Iwwxwcw, S., TATSUKI, T. and Suzutü, T. (1976) Snake venom proteinase inhibitors-III. Isolation of five polypeptide inhibitors from the venom of Hemachatus haemachatus (ringhaLs cobra) and Naja nivea (Cape cobra) and the complete amino acid sequence of two of them . J. Biochem., (Tokyo) 79, 559. Iwwxwcw, S., WALLEN, P., Gxörrowm,, N. J., HENSCxEN, A. and B.ot~wcx, B. (1969) On the primary structure of human fibrinogen . Eur. J. Biochem. 8, 189. KASSELL, B. and Lwsxowsxi, M. (1965) The basic trypsin inhibitor of bovine pancreas-V . The disulßde linkages. Biochem. biophys. Res. Common . Z0, 463. KwuL, P. N. and SirmaRnswivx, C. J. (Eds ., 1978) Drugs and Food from the Sea. Myth and Reality. Univ, of Oklahoma, Norman, Oklahoma . LOWRY, O. H., R03EHAOUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent . J. biol. Chem. 193, 265. Mwxnv~rrt, G. V. (1965) The action of phospholipase A on lipoproteins . Biochim . biophys. Acts. 98, 554. MUSCATINE, L. and LENHOFF, H. M. (Eds., 1974) Coelenterare biology. Reviews and New Perspectives . Academic Press, New York . NoR~rox, T. R., KASHIWAaI, M. and $HIBATA, S. (1978) Anthopleurin A, B and C, cardiotonic polypeptides from the sea anemones Anthopleura xanthogrammica (Brandt) and A. elegantlssima (Brandt) . In : Drugs acid Foodfrom the Sea, p. 37 (ICwur., P. N. and $IIYD&RMANN, C. J., Eds.). Norman, Oklahoma. POOR, N. and ZLOrxu~r, E. (1975) On the ichthyotoxic and hemolytic action of the skin secretion of the flatfish Pardachirus marmoratus (Soleidae). Toxicon 13, 227. RATHMAYER, W., JESSex, B. and BERE33, L. (1975) Effects of toxins of sea anemones on neuromuscular transmission . Naturwissenschaften 62, 538. Sawrmo, B. I. (1968) Purification of atoxin from the tentacles of the anemone Condylactis gigantea . Toxicon 5, 253. SHIHATA, S., NORTON, T. R., Izurn, T., Mw~rsvo, T. and Kw~rsuxr, S. (1976) A polypeptide (AP-A) from sea anemone (Anthopleura xanthogrammica) with potent positive inotropic action . J. Pharmac. exp. Ther. 199, 298. SIMP$ON, R. J., NEUBEROER, M. R. and Lru, T. Y. (1976) Complete amino acid analysis of proteins from a single hydrolysate. J. biol. Chem. 251, 1936. TuR~wewTV, P., SHIHATA, S., NoRTOx, T. R. and Kwsfnwwci, M. (1973) A possible mechanism of action of a central stimulant substance isolated from the sea anemone Stoichactis kenti. Eur. J. Pharmac. 24, 310. Wurnet~x, G., BeRESS, L., MACHLEIDT, W. and FRrrz, H. (1976) Broad-specific inhibitors from sea ane mones. In : Methods fn Enzymology, Vol. 45, p. 881 (LoRwtvn, L., Ed.). London : Academic Press.