Toxfcon, 1977, VoL 13, pp. 38393 . PerBrmon Pros .

Printed io t3reat Britain

ISOLATION AND CHARACTERIZATION OF A LETHAL MYOTOXIC PHOSPHOLIPASE A FROM THE VENOM OF THE COMMON SEA SNAKE ENHYDRINA SCHISTOSA CAUSING MYOGLOBINURIA IN MICE
JaN Foi-u.n~ax and Dev>D EAxEx Institute of Biochemistry, Uppsala University, Box 376, 5-751 23 Uppsala, Sweden
(Accepted jor publtcatlon ZO January 1977)

J. Fot~aswx and D. Fr+xEa . Isolation and characterization of a lethal myotoxic phospholipase A from the venom of the common sea snake Enhydrtna schistose causing myoglobinuria in mice . Toxicon 15, 385-393, 1977.-A strongly ~yotoxic phospholipase A was isolated from the venom of F,nhydrlna schistose by gel filtration on Sephadex G-73 and ion exchange chromatography on Bio-Rex 70. The myotoxin is a basic protein containing 120 amino acid residues and seven disulfide bridgos. The formula weight is 13,500. The mlease of myoglobin diagnostic of muscle necrosis was monitored by placing iltjected mice on sheets of white paper. Affected mice stained the paper red. The myotoxin has an acute ~so of 110 uglkg and causes myoglobinuria at doses down to 30 kg/kg. The molecule lacks tryptophan, indicating that this amino acid is not required either for toxicity or catalytic activity. A singlo histidine residue wasmodified by reaction withp-bromophenacyl bromide, resulting in > 99 loss of enzymatic and toxic activities . Tho N-terminal sequence shows homology with nontoxic and neurotoxic phospholipases AZ of vertebrate origin . The myotoxicity may be of greater clinical importance than the curariminletic toxins that have received so much attention heretofore.

Sam. sxnxFS abound in the warmer Asian waters, and although bathers are rarely bitten the snakes are an occupational hazard to fishermen (BAx~, 1963, 1968 ; R, 1961a). The common sea snake, Enhydrina schistose Daudin is the most abundant, most poisonous, and accounts for most sea snake bites involving humans (R>uD,1961a) . Theearliest andmost conspicuous symptom ofE. schistose bite in man is generalized muscle pain followed 3-6 hr later by myoglobinuria, indicative ofextensive muscular damage (REm, 1961a, b; M~tsDErt and ItEID, 1961). In earlier papers from this laboratory, KARi.ssoN et al. (1972) reported that about 60 of the protein content of Enhydrina schistose venom consisted of rura_rimimetic postsynaptic neurotoxins having t.nao doses below 10011g/kg mouse ; the value given for the whole dried venom by CARSY and Wxioi-rr (1960). FYtYxt urm et al. (1972) reported the amino acid sequences ofthe two principle neurotoxins which together accounted for about 40 ~ of the venom protein . Although the content of post-synaptic neurotoxins is adequate to account for the i.n6o dose of the venom in mice, curarimimetic activity cannot be responsible for the clinical picture in human victims . HeRxts et al. (197 found that notexin from the Australian tiger snake (Notechis s. scutatus) induced damage to skeletal muscle. Furthermore, anti tiger snake serum gives good pare-specific protection against sea snake venom (MuvTON, 1967) . These two obser vations suggested that the myonecrotic factor of Enhydrina schistose venom might be a structural homolog of notexin, and this is borne out by the results presented in this paper.
385

INTRODUCITON

386

JAN FOHLMAN and DAVID FAKER MATgRiAi C AND METHODS

Crude venom Dessicated Enirydrina schistose venom was obtained from Mrs . Oo Sooi Lix, Department of Pharmacology, University of Malaya, Kuala Lumpur 22-11, Malaysia . Gel filtration Two grams of crude venom was dissolved in 10 ml of 0 " 2 M ammonium acetate with gentle shaking on a Whirhnixer. The foam was broken with the tip of a toothpick moistened in 1-octanol, insoluble debris was removed by centrifugation at 1000g for 5 min followed by ZO min at 20,000 x g, and the clear, ochre supernatant solution was gel filtered on Sephadex G-75 in 0~2 M ammonium acetate. Ion exchange chromatography About 250 mg of eeze-dried protein from the myotoxic gel filtration fraction VI was dissolved in 7 ml of 005 M ammonium acetate and chromatographed on a column of Bio-Rex 70, -400 mesh, equilibrated with 0 " 2 M ammonium acetate at pH 7" 5 as described by Kwxissox et al. (1971) . Prior to applying the sample about 100 ml of 005 M ammonium acetate was pumped into the column. The column was eluted with a 2-1 concave gradient of 005 M vs 1 M ammonium acetate formed by 2 cylinders having a surface area ratio of 1 : 2, the larger being the mixing/output cylinder containing initially the weaker buffer . Assay for myoglobinurla Myotoxicity was revealed by the appearance of myoglobin in the urine . Injected mice were placed on white filter paper in separate cages . Control urine was colourless or faintly yellow. Severely intoxicated mice stained the paper red . The urine from these animals was eluted from the paper with physiological saline . No erythrocytes were observed and the presence of myoglobin rather than hemoglobin in the extracts was confirmed by running a spectrum, since the spectra of the two proteins are distinctly different . Lethality assay ~so was determined by i.v . administration using 4 mice at each dose level . The toxin concentration was measured spectrophotometrically using tho molar absorptivity value determined in conjunction with amino acid analysis . Phospholipase assay Phospholipase activity was measured using a modification of the method of DE Hwws et al . (1971) One part egg yolk was mixed with 1 part 18 mM CaCI, and 1 part 8" 1 mM sodium deoxycholate . The pH was adjusted to 8 " 0 with NaOH . Two ml of this solution was titrated at room temperature with 001 M NaOH in a pH-stet under nitrogen after addition of a known amount of enzyme . The liberation of free fatty acid was computed as pmole/min/mg . The initial velocity was measured as the initial slope, since the rate was linear during the first three min at the enzyme concentrations used . Modif :catton reaction The myotoxin was modified with p-bromophenacyl bromide as described by VoLwsiuc et al . (1974) and by Hwt reRT et al. (1976) for porcine pancreatic phospholipase AZ and notexin, respectively . The protein (0"92 umole) was dissolved in 2 ml 0" 1 M cacodylate buffer, pH 6"0, containing 0~1 M NaCI . p-Bromophenacyl bromide (5 umole) was added and allowed to react for 18 hr at 30C . The reaction mixture was gel filtered on a short column of Sephadex G-25 and the void peak was collected and lyophilized . A spectrum was run in conjunction with amino acid analysis to determine the molar absorptivity, and thereby, the degree of reaction . Amino acid analysis Lyophilized protein samples were hydrolyzed for 24 and 72 hr with 6 N HCl containing 10 mg/ml reagent grade phenol in thoroughly evacuated tubes at 110C . Total half-cystine and methionine were determined as cysteic acid and methionine sulphone, respectively, after performic acid oxidation . Tryptophan was estimated from ultraviolet spectra run in conjunction with amino acid analysis, using molar absorptivity values of 5554 for tryptophan, 1260 for tyrosine and 150 for disulfide bridges at 278 nm . Preparation of reduced and S-carboxymethylated derivatives Protein (2 umole) was dissolved in 2 ml of 6 M guanidine hydrochloride containing 086 M Tris HCI, pH 8 " 6 and 003 ~ EDTA . Dithioerythritol (ZZ mg) was added and the reduction was allowed to proceed under nitrogen at room temperature for 6 hr. Then 25 gl of a solution containing 2 " 3 mg/m13H iodoacetate (specific activity 90 mCi/mmole) was added and allowed to react for 10 min, after which complete alkylation was accomplished by the addition of 61 mg "cold" sodium iodoacetate . The radioactive label was introduced to facilitate eventual peptide isolation and identification of cysteine residues in the sequence work now in progress . N-terminal sequence Edman degradation of the reduced and S-carboxymethylated myotoxin was done by the direct phenylisothiocyanate method as described by Iwwxwaw et al. (1969) and EnhrwN and HexscaEx (1975) . After

Sea Snake Myotoxin

387

spxtrophotometric determination of the concentration, S nmole portions of the ethylacetate~olublo PTHderivatives were identified by thin layer chromatography on silica gel plates (Merck Fertigplatten FJ in solvent V (JEPPSON and SIOQuISr,1967) and solvents II and III(BRENNER et a1 .,1962). The spots were located visually under ultraviolet (254 nm) illumination . The aqueous phases ware examined for PTH-arginine and PTH-histidine by paper electrophoresis at 440 V for 2 hr at pH 6 " 5 in sodium phosphate buffer containing 0"015 M Na,HPO,, 0" 03 M NaH,PO 1 g of disodium 13DTA and S g of soluble starch per liter. PTH-derivatives were visualized as white spots on a wffeo-coloured background by means of tho iodine azidoreagent (EuMAN and Hi;NSCIIert,197S). RESULTS Isolation of myotoxin YI : S

The elution pattern of Falltydrina schistose venom protein on Sephadex G-75 is shown in Fig. 1 (insert) . A retarded peak of nucleosides is not shown. Nearly all of the protein constituents elute in two size fractions : fraction VII representing about 70 ~ of the total protein and a molecular weight of 7000, and fraction VI containing 20 ~ of the total protein and corresponding to a molecular weight of about 14,000 . Fraction VII consists mainly of curarimimetic neurotoxins (KARlssox et al., 1972). Fraction VI showed phospholipase activity, was lethal to mice, and produced myoglobinuria . It was further fractionated by ion exchange chromatography on the polycarboxylic resin Bio-Rex 70 . Seven peaks were observed, as shown in Fig. 1, and accounted for 6, 2, 1, 3, 7, 1 and 1 ~ respectively, of the total venom protein. The unretarded peak VI : 1 produced no symptoms in mice at a dose level of 500 Ftg/kg. VI : 5 was highly lethal and produced myoglobinuria, and was therefore studied in more detail . The other peaks were not assayed. Characterization of myotoxin VI : S The amino acid composition (Table 1) is very close to integral for all amino acids except Asn and Leu, perhaps indicating a discrete Asn-Lee substitution. The postsynaptic toxins 4
ao

zo H

The 32 x 30~:m ion exchange column was packed with Bio-Rex 70 as described in Methods. The main myotoxic pock emerges at an approximate ammonium acetate wncentration of 0"S M. x is expressed in m/~l'1 x cm'1. Fraction volume =9 ml.Insert near top of figure shows the protein distribution obtained in a separation of 2 g credo venom on a 3 " 2 x 92 " S~m column of Sephadex G-75 . Fraction VI corresponds to a MW of 14,000 and VII about 7000.

FIO. 1 . ION EXCHANGE CHOMATOORAPHY (1 b) OF 3SO mg OF FRACITON VI (~~PHOSPFIOLiPASE FRACITOI~~ FROM TAE INITIAL OEL FII.TRATION STEP SHOWN IN TOP INSERT.

38 8

JAN FOHLMAN and DAVID ERKER

TAHLE 1 . AMnvO ACID COMFO~IION OF FJJ%lyLlrlna

Residuo Asx Thr Ser Gk Pro Gly Ala Cys' Valt Met IIet Leu Tyr Phe His Lys Trp Arg No . of residues Formula weight Molar absorption (276 nm) As7e (1 mg/m1)

VI :S

SCIllStOJa MYOT'OXIN Integer 21 4 S 6 3 9 10 14 6 2 3 4 12 2 2 10 0 7 120 13,500 16,900 l

Mole ratio 20" 6 3 " 91 5 " 24 5 " 90 2" 97 9 " 09 9 " 97 14 " 1 5 "92 1 "96 314 443 11 "7 1 ~91 2"00 10 "0 0 6"95

'Average of values fr S~arboxyme thykysteine and cysteic acid. t72 hr value only.

and 5 from the same venom diered by a discrete Pro-Ser substitution (FxYxLUxn et al., 1972). The spectrum has a maximum at 276 nm, indicating a high content of tyrosine (12 from amino acid analysis) and this, together with the molar absorptivity value 16,900, is not compatible with the presence of any tryptophan. The myotoxin contains no amino sugars. N-terminal analysis Eleven turns of Edman-degradation on the reduced and S-carboxy-methylated derivative established the N-terminal sequence Asn-Leu-Val-Gln-Phe-Ser-Tyr-Val-IIe-Gln-Gj~s, which shows close homology with many other phospholipases AE as illustrated in Fig. 2. On the basis of the obvious homology we infer that the 14 cysteine residues are connected by 7 disulfide bridges . Toxicity At doses above 100 Ftg/kg the mice seem to die from respiratory failure, the symptomaMyotaxin Notexin N . nig . Plase Aan-Leu-Val-Gln-Phe-Ser-Tyr-Val-Ile-Gln-CysAsn-Leu-Val-Gln-F ;ie-Ser-Tyr-Leu-Ile-Gln-CysAsn-Leu-Tyr-Gln-Phe-Lys-Asn-Met-Ile-His-Cys Asn-Leu-Leu-Gln-Phe-Gly-Phe-Met-Ile-Arg-Cys-

Taipoxin a Pork

Taipoxin ~

p . Plase

Asn-Leu-Val-Gln-Phe-Gly-Phe-Met-Ile-Gln-CysAla-Leu-Trp-Gln-Phe-Arg-Ser-Met-Ile- Lys -Cys-

Fra. 2. N-TERMINAL HOMOLOGY AMONG SOME TOXIC AND NON-TOXIC FHOSPHOLIPASFS . Notexin is from Australian tiger snake (Notechis s. scutatus, HALPERT and EAICER, 1975), the basic phospholipase from African spitting cobra (NaJa nlgricollls, OHII)AIRO et al., to be published), taipoxin from the Australian taipan (Oxyuranty s. acutellatw, FOHLMAN et al., 197 and pork pancreatic phospholipase (DE HAAS et al., 1970).

Sea Snake Myotoxin

38 9

tology closely mimicking envenomation with presynaptic toxins (like taipoxin or notexin) . If the mice do not die within 12 hr they either recover or go into a state of catabolism, apparently using up all energy stores. They lose weight, move about shakily and die on the 4th or 5th day in an emaciated state. Probably much of the skeletal muscle mass is severely degraded by this stage. The toxicity thus involved two symptoms which are apparently due to quite different pharmacological and pathogenic mechanisms . We have accordingly determined two 1.Dso 'S, one at 110 LIgJkg for the fast-ensuing paralytic death and one at 40 FtgJkg for the wasting death. The clinical importance of this finding is evident. Myotoxicity The myotoxicity has so far been followed only by the simple assay for muscle-destruction manifested by the appearance in the serum of myoglobin, which is rapidly cleared through the kidneys and is easily detected by the colour of the urine. Myoglobinuria appears at dose levels above about 30 llgJkg, which means that the toxin is extremely toxic for skeletal muscle . The 1.n6o for intraperitoneal injection is about ten times that obtained for intravenous injection. activity i:n the presence of sodium deoxycholate a catalytic activity of 200 lTmoleJminJmg was determined at room temperature using egg yolk as substrate. The substrate specificity was not investigated .
Phospholipase Modification reaction

The myotoxic phospholipase reacts with p-bromophenacyl bromide as evidenced by the 2~5-fold increase in the molar absorptivity at 270 nm (Table 2). This, together with disappearance of one histidine residue (as determined by amino acid analysis) suggests that the modification is the same as was observed with the pancreatic phospholipase (VOLWERK et a1.,1974) and notexin (IIALPERT et al., 197 . The pAs,n value is somewhat higher than that reported for the protein-conjugated reagent in the latter reference. After modification the myotoxin produced no sign of myoglobinuria or any other distress in mice at doses corresponding to 50 x LDS o ofthe native protein. The phospholipase activity was 1 lTmole/minJmg, or 0~5 ~ that of the virgin protein. By these measures both the lethality and the enzymatic activity were decreased by 99 ~. The parallelism between the myotoxic, neurotoxic, and catalytic activities is discussed below.
TAHIE i.

SOe~ CHARACiEItL4ITC8

OF NATIVE AND P-HROMOFHENACYL HROI~E-REACTED bIYOTOXIN

Native No . of histidine residues (amino acid analysis) 200 Molar extinction coefficient (270 nm) 13,600 p-Bromophenacyl bromide residues/mole protein` 0 Phospholipase activity (umole fatty acid/mg x min, see Methods) 200 110 ~so (ug/kg) : Acute Long term Smallest myoglobinuric dose 40 30
(HALPERT Ot si .,

Modified 1 " 09 34,600 1 "2 1 > 5000 >5000

`e.,o ~ (PBP-cor{jugate) 17,000

1976).

390

JAN FOHLMAN and DAVID ERKER DISCUSSION

Phospholipases are almost invariably present in snake venoms (Ivhs, 1970) . Their function must be considered to be originally digestive, since these enzymes are a common feature ofthe alimentary canal. In recent years, however, it has become evident that phospholipases Ae have evolved quite specialized toxic properties in snake venoms, as exemplified by the neurotoxicity ofthe structural homologs notexin (HALPERT and Enxm, 1975), talpoxin (FoxLntAN et al., 197, crotoxin (Bx~s~urr et al., 1975), and probably (3bungarotoxin . H,~uus et al. (1975) showed that notexin also has myotoxic properties . Apparently the Enhydrina schistosa myotoxin VI : 5 also has three biological activities, and the relationships among them remain to be elucidated . Membrane specificity Phospholipases A hydrolyze organized lipid much more rapidly than they attack monomeric substrate (e.g. SLOTBOOM and DE Haas, 1975). The leakage of myoglobin from the myofibrils logically implies some kind of membrane damage . The membrane function has been shown to be impaired after poisoning with the presynaptic neurotoxins talpoxin and notexin (CULL-CANDY et al., 1976). Interaction with organized lipid (e,g. membranes or substrate micelles) is thus a common feature of the two quite different toxic activities and the catalytic activity as well. Initially we hoped to find an exclusively myotoxic phospholipase, but have instead found a molecule with a different myotoxic/neurotoxic ratio than notexin. The N-terminal region appears to play an essential role in the formation ofthe interface recogntion site (VAN D.~vt-MtExns et al., 1975), but as shown in Fig. 2 there is no striking dissimilarity among six different phospholipases in this region . Thus the cell specificity must reside somewhere else in the molecule . The apparent preference for different cell-membranes must have some structural basis. We can think of three possibilities : (1) Different packing of phospholipids in the different cell membranes . If catalysis is involved in the biological effects the surface tension could be crucial (ZwaaL et al., 1975 ; DEAL et al., 197 . (2) Different cell membranes may have different 3-sn-phosphoglycerides, i.e. differences in the long~hain fatty acid esters or the alcohol part. This could account for the celldirected specificity and should be experimentally tested using different synthetic derivatives. (3) A structural or functional protein in the membrane might be the main target of the attack . It might be a permease or an enzyme the proper function ofwhich is dependent on the integrity of the surrounding lipid bilayer . The loss of both the catalytic and the pathological activities upon chemical modification with p-bromophenacyl bromide cannot alone suffice as proof that the phospholipase activity is directly involved in the toxic action(s). Recent crystallographic work with the pancreatic prephospholipase shows histidine and tyrosine in the catalytic cleft. In the neighbourhood is an electron density which could be a Ca ion, perhaps coordinated by an aspartic acid and an asparagine residue (DxeN~rx et al., 1976) . The introduction of the rather bulky p-bromophenacyl residue severely hampers the ability to bind calcium and sterically disturbs the catalytic site. Apparently the integrity of the catalytic cleft is important for both the neuro- and myotoxic activities, whether catalysis is involved or not. Since the myotoxin with its comparatively high specific phospholipase activity is devoid of tryptophan this amino acid can have no important role in the hydrolysis ofphosphoglycerides . It is hard to define simple end-points for the LDbo determinations since the acute and

Sea Snake Myotoxin

39 1

chronic effects overlap. When there are long-term effects on locomotor organs one must also take into account starvation or dehydration. That in fact two different activities are present must be further investigated by electrophysiological and histological techniques. Taipoxin is a poorer myotoxin than notexin but is much more potent presynaptically (Fofu.Max et al., 1976). The Enhydrna schistosa myotoxin is the poorest pre-synaptic neurotoxin of the three, but is the only one of them that produces muscle degeneration upon intravenous administration (Hnnx>s, personal communication) . However, here we have the complication that the dose of notexin required to produce muscle degeneration upon intravenous administration might be several times the lethal paralytic dose. The animal thus expires before the symptoms of myotoxicity appear. Pathogenic sole of the myotoxic phosphollpases Whether sea snake poisoning is primarily myo- or neurotoxic has been the subject of some discussion (R~, 1961b; MnRSnsx and R>~, 1961). Biochemical separation methods have demonstrated the presence of both myotoxins and neurotoxins. The pathogenic balance between the two remains to be elucidated and probably depends on the species of the victim. For example, man might be considerably more sensitive to the myotoxin (or alternatively, less sensitive to the curarimimetic toxins) than are mice. The following facts should be considered : (1) Mice injected with the curarimimetic post-synaptic neurotoxins die within 2-4 hr or recover completely within 12 hr. (2) No improvement in victims of sea snake bite is observed after neostigmine administration (lt~, 1961a) . However, this treatment might not be as diagnostic as it at first seemed, since it is difficult to prove that neostigmine (or any acetylcholine-esterase inhibitor) should have significant clinical value in humans, although claims have been made (Bnt~~ et al., 1974). (3) Mortality among sea snake victims is as high as 17 ~ of the cases seen at hospital, and 27 ~ of the deaths occur after 2 days. The average death time is 30 hr (REm, 1961a, 25 cases) . (4) The lethal dose for one adult human is estimated at 1~5 mg, or about 10 ~ of the total venom yield (Muv~rox and Mnv~rox, 1969). (~ The lethal dose for the venom of the Asian cobra, Naja raja, is estimated at 15-20 mg, again about 10~ of the total gland content. The total amount of curarimimetic neurotoxins injected is perhaps 5 times that injected in a sea snake bite but > 95 ~ of the victims recover (Mnaz~ox and MtrrroN, 1969). Humans usually die within 8 hr (Itt?ro, 1964). These observations suggest that something other than (or in addition to) curariform neurotoxins is responsible for the ultimate fatal outcome or m im;ng effects of sea snake bite. We suggest that this may be the basic myotoxic phospholipase(s) . The mode of action of the myotoxins is not known. Neither notexin nor taipoxin exhibit myotoxicity in vitro, although in vivo both are highly potent (Hnxxrs, personal communication) . Indirect evidence suggests that muscle damage is followed by the invasion ofphagocytic cells, the invading cells being rich in peptide-hydrolase enzymes.The "trigger" stimulating the invasion of these cells may be some factor released by the damaged muscle fibre membrane, or compounds released by other cells (e.g. mast cells) that are also damaged by the toxin (Pi.usxnL et al., in press) . Preliminary evidence (H~xxis and Joxrrsox, personal communication) suggests that muscle damage following sea-snake myotoxins shares all the features so far described for notexin (Hnxxis et al., 1975). The modified myotoxin should be an excellent toxoid for the production of antisera, since very high doses can be injected to obtain a good response without killing the animal .

392

JAN FOHLMAN and DAVID BAKER

This has alreadybeen done with the p-bromophdenacyl bromide modified taipoxin (RAMLAU et al., to be published). The reason for the good paraspecific protection by tiger snake antivenom now seems apparent, since notexin and the myotoxin are similar molecules. This also indicates the pathogenic importance of the myotoxin .
Acknowledgements-We aregreatly indebted to Mrs. Oo Sooz Lrnr, Department of Pharmacology, University of Malaya, for supplying the crude Erhydrina schistose venom . We thank Miss MARIAxxE NoRnlnYC~ for enthusiastic help in the isolation of the myotoxin, and value the helpful criticisms of Dr. EvERZ KARl ssort. The investigation was supported by the Swedish Natural Science Research Council. dnr 2859-008 . REFERENCES

BANERJEE, R. N., SAExI, A. L. and CaAClco, K. A. (1974) Neostigmine in the treatment of Elapidae bite. 4th Int. Synrp. on Amiral, Plant and Microbial Toxins, Tokyo, 8-13 September, 1974 . BARME, M. (1963) Venomous sea snakes of Viet Nam and their venons. In : Venomous and Poisonous AnirnaLs and Noxious Plants of the Pacifre Region, p. 373, (KEEGAx, H. L. and MACFARLAIaE, W. V.,Eds.l . Oxford : Pergamon Press . BARME, M. (1968) Venomous sea snakes (Hydrophdae). In : Venomous Animals oral Their Venons, Vol. I, p. 285, (BUCHERL, W., BucxI.EV, E. and DEULOFEU, V., Eds.) . New York : Academic Press. BREITHAUPT, H., OMORIzSATOFI, T. and LANG, J. (1975) Isolation and characterization of three phospholipases from the crotoxin complex. Biochim. biophys. Acta 403, 355. BRENNER, M., NIEDERWEI3ER, A. and PATAKI, G. (1962) In : Diaurschichtchromatographie, p. 443, (SzAm., E., Ed .). West Berlin : Springer . CARET, J. C. and WRIGHT, E. A. (1960) The toxicity and immunological properties of some sea-snake venons with particular reference to that of F .nhydrina schistose. Trans. R. Sce. trop. Med. Hyg. 54, 50 . CLLrCANDY, S. C., FOErc.MAN, J., GUSTAV930N, D ., LuLL.MANN-RAUCH, R. and THESLEFF, S. (1976) The effects of taipoxin and notexin on the function and fine structure of the marine neuromuscular junction . Neuroscience 1, 175. DAM-MmRAS, M. C. E. vAN, SLOZaooM, A. J., PIEZERSON, W . A. and HAAS, G. H. DE (1975) The interaction of phospholipase A, with micellar interfaces. The role of the N-terminal region . Biochemistry 14, 5387. . S. M., ZWAAL, R. F. A., ROEIAFSEN, B. and DEENEN, L. L. M. vnN DEMEL, R. A., GEURTS vAN KESSEI., W (1975) Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers. Biochim . blophys . Acta 406, 97 . DRENTFi, J., ENZINC, C., KAtx, K. and VESSIES, J. (1976) Structure of porcine pancreatic prephospholipase A, . Nature 264, 373. EUMAN, P. and HEN9CHEN, A. (1975) Sequence determination . In : Protein Sequence Determination, p. 232, (NEEDLEMAN, S. B., Ed .) . West Berlin : Springer . FOHLMAN, J., EAxER, D., KARLSSON, E. and TIIESLEPF, S. (1976) Taipoxin, an extremely potent presynaptic neurotoxin from the venom of the Australian snake Taipan (Oxyurartus s. scutellatus). Eur. J. Blochem. 68, 457 . FRYKL.LiND, L., EARER, D. and KARL490N, E. (1972) Amino acid sequences of the two principal neurotoxins of Enhydrirra schistose venom. Biochemistry 11, 4633 . HAAS, G. H. DE, Sl.ozeooM, A. J., BONSEN, P. P. M., DEENEN, L. L. M. vAN, CHAROUX, S., PutcseRVER, A. and DE3NEULLE, P. (1970) Studies on phospholipase A and its zymogen from porcine pancreas-I . The complete amino acid sequence . Biochim. biophys. Acta 221, 31 . HALPERT, J. and EAR .ER, D. (1975) Amino acid sequence of a presynaptic neurotoxin from the venom of Notechts scutatus scutatus (Australian Tiger snake) . J. biol. Chem . 250, 6990 . HALPERT, J., EAKER, D. and KARLS.40N, E. (1976) The role of phospholipase activity in the action of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian Tiger snake) . FEBS Lett . 61, 72 . HARRIS, J. B., JOHNSON, M. A. and KARL430N, E. (1975) Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of the Australian Tiger snake (Notechts s. scutatus). Cllr. Exp. Pharmacol. Physiol. 2, 383. IWANACiA, S., WALIBN,P., GRSNDAL, N. J., HENSCHEN, A. and BLOMBACK, B. (1969) On the primarystructure of human fibrinogen . Eur. J. Bloehem. 8, 189. JErrsoN, J. O. and SroQulsr, J. (1967) Thin-layer chromatography of PTH-amino acids. Analyt . Biochem.l8, 264. KARL430N, E., ARNHERG, H. and EARER, D. (1971) Isolation of the principal neurotoxins of two NaJa naJa subspecies . Ear. J. Blochem. 21, 1 . KARL.430N, E., EAxER, D., FRYKLUND, L. and KADIN, S. (1972) Chromatographic separation of F .rhydrlna schistose (common sea snake) venom and the characterization of two principal neurotoxins . Biochemistry 11, 4628 . MAR3DEN, A. T. H. and RE7, H. A. (1961) Pathology of sea-snake poisoning. Br. Med. J. 1, 1290 .

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393

M$es, D. (1970) A comparative study of enzyme activities in snake venoms . Int.1. Biochem. l, 335. Mcxrox, S. A. (1967) Paraspocific protection by elapid and sea snake antivenins . Toxicon S, 47. Mrxzox, S. A. and Mixzox, M. R. (1969) Venomous Reptiles. New York ; Charles Scribner . Pt .usg.~ ., M. G., H~xnis, J. B., PExxixa~rox, R. J. and EucEx, D. (1976) Some biochemical responses of rat skeletal muscle to a single subcutaneous injection of a toxin (Notexin) isolated from the venom of the Australian Tiger snake (Notechis s. seutatus). CJtn. Exp. Pharnracol . Physiol. To be published. REro, H. A. (1961a) Diagnosis, prognosis and treatment of sea-snake bite . Lancet , 399. REID, H. A. (19616) Myoglobinuria and sea-snake bite poisoning. Br. Med. l. l, 1284 . R$m, H. A. (1964) Cobra bitty. Br . Med.1. 2, 540. Sr ateooM, A. J. and Haves, G. H. na (1975) Specific transformations at the N-terminal region of phospholipase A, . Biochemistry 14, 5394 . VOiWERIC, J. J., Pt$rexsox, W. A. and Hoes, G. H. DE (1974) Histidine at the active site of phospholipase A, . Biochemistry 13, 1446 . Zweet., R. F. A., Rortorssx, B., Coxroxias, P. and Draxax, L. L. M. vex (1975) Organization of phaspholipids in human red cell membranes as detected by the action of various purified phospholipases . Btochim. biophys. Acta 406, 83