Separation and partial characterization of smooth muscle contractile material in the venom of the scorpion Heterometrus bengalensis

Toxicon, Vol . 21, No . 3, pp . 353-361 . 1983 . Printed in Great Britain .

0041-0101/83503 .00 + .00 © 1983 Peraamon Press Ltd.

SEPARATION AND PARTIAL CHARACTERIZATION OF SMOOTH MUSCLE CONTRACTILE MATERIAL IN THE VENOM OF THE SCORPION HETEROMETRUS BENGALENSIS PARITOSH

K. KAR, BANDITA SARANGI, APARNA DATTA, ANTONY GOMES and S. C . LAHIRI*

Department of Pharmacology, N. R. S. Medical College, Calcutta 700 014, India (Accepted for publication 10 August 1982)

KAR, B . SARANGI, A. DATTA, A. GomEs and S. C . LAHIRI . Separation and partial characterization of smooth muscle contractile material in the venom of the scorpion Heterometrus bengalensis. Toxicon 21, 353 - 361, 1983 . - A smooth muscle contractile material was separated from crude venom of the scorpion Heterometrus bengalensis (found in Eastern India) by solvent extraction, gel filtration and thin layer chromatography . Smooth muscle contractile material could be extracted, in descending order of efficiency, with methanol, butanol, ethanol and acetone. The contractile material separated by gel filtration (Sephadex G-25) when further extracted, using the Folch procedure, showed a single spot in thin layer chromatography with one solvent system . Rechromatography of an eluate from this spot with another solvent system resolved it into three spots (SLI, SL2 and SL3, the mixture being designated as Substance L) which could be visualized either with iodine vapour, ninhydrin or molybdenum reagent. Eluates from the three spots contracted guinea-pig ileum which had been pretreated with antagonists of ACh, histamine, 5-HT and prostaglandins . Substance L and its fractions (SLI, SL2 and SL3) contain inorganic phosphorus, amino nitrogen and amino sugar, which point to the likelihood of their being glycophosphatides .

P. K.

INTRODUCTION

WHILE some information is available about smooth muscle stimulating activity of scorpion venoms (DEL POZO, 1956; DINIZ and GONCALVES, 1956; DINIZ and VALERI, 1959 ; TAZIEFF-DEPIERRE, 1968, 1972; DINIZ, 1971 ; ISMAIL et al ., 1973 ; CUNHA-MELO et al., 1973 ; ISMAIL et al., 1974) there are no reports using the venom of Heterometrus

bengalensis, which is common in Eastern India. Nag Choudhuri and Lahiri (unpublished observations) observed that H. bengalensis venom contracted smooth muscles even in the presence of antagonists of ACh, histamine and prostaglandin. This suggested the presence of a substance(s) other than the agonists mentioned above. This surmise was further strengthened when thin layer chromatography of venom elicited a spot which was distinct from spots produced by authentic ACh, histamine, 5-hydroxytyptamine and prostaglandin . The present investigation was undertaken to separate and characterise the smooth muscle contractile substance(s) from the crude venom.

*To whom correspondence should be addressed at the Department of Pharmacology, N.R .S . Medical College, 81 Rash Behari Avenue, Calcutta-26, India. 353

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P. K. KAR et al . MATERIALS AND METHODS

Collection of venom The telson of scorpions, held in a modified mouse trap (WHITTERMORE, 1963), was stimulated with square wave stimuli (2-4 volt, 0.5 msec/10 Hz). Venom, obtained once in 3-4 weeks, was pooled and placed in a vacuum desiccator over fused calcium chloride . Dry venom was stored at -20°C until use. Immediately before use crude venom was diluted with the appropriate physiological solution .

Single and multiple solvent extractions

The venom was homogenised with a single solvent (chloroform, methanol, ethanol, butanol, ether or acetone), shaken for 30 min and centrifuged (1800 d x 15 min), the supernatant being dried in a partial vacuum . Smooth muscle contractile activity was estimated on atropine -mepyramine-cyproheptadine-treated (AMCtreated) isolated guinea-pig ileum. Gel-filtrated material obtained from dried crude venom was extracted according to the procedure of FOLCH (1957), vacuum-dried and kept under a nitrogen atmosphere at -20°C. In some experiments the extraction procedure of BLIGH and DYER (1959) was tried as an alternative procedure, however, it gave a slightly lower yield. The dried material was stored in chloroform solution, vacuum-dried and reconstituted just before use . Gelfiltration and adsorption chromatography Gel filtration was carved out on a Sephadex G-25 column (15 x 300 nlm) with a flow rate of 10-15 ml/hr. The column was eluted with 0.05 M phosphate buffer, pH 7.2, at 4°C. Aliquots of a constant volume (3 ml) were collected . Various fractions were tested for contractile activity on AMC-treated guinea-pig ileum. A sintered column (15 x 100 mm) was used for adsorption chromatography, with a flow rate of 0.2-0 .3 ml/min, at 4°C. The eluate was vacuum-dried and tested for activity .

Thin

layer chromatography Silica gel G type 60-coated (250 Nm) plates were prepared (STAHL, 1965). The test substances were applied as a single spot or a 5 cm strip. The material was allowed to ascend to a height of 16 cm . The solvents used were : isopropanol - 0. IN HCL (7 :3); isopropanol - water (7 : 3) ; chloroform -methanol - water (60 : 35 : 8) (Solvent System A); chloroform-methanol-acetic acid-water (100:50:14:6) (Solvent System B) ; chloroform-methanol-water (10:5 :1); chloroform-methanol-acetic acid (12 : 8 : 1) . Chromatograms were developed with : iodine vapour ; ninhydrin in acetone (0.2% solution) ; ninhydrin in n-butyl alcohol (water-saturated, 0.5% solution) ; molybdenum blue reagent. Other methods Lipid phosphorus was determined according to the modified method of BARTLETT (1959) described originally by FLsKE and SUBBA Row (1925). Protein was estimated by the procedure of LOWRY et al. (1951) . Amino nitrogen of the component was estimated as described by ROSEN (1957) . Carbohydrate content of the contractile material was estimated by the anthrone technique (TREVELYAN and HARRISON, 1951), Molisch and Benedict tests . Parallel bioassay of smooth muscle contractile material against prostaglandin E E, and F,, was carried out according to the method of GADDUM (1955) . Stability studies After keeping in a boiling water bath for 10 min, samples were cooled to room temperature and corrected for evaporation loss, if any. Activity was compared with that of an untreated control. In some cases the material was acidified with 0.5 ml of 4 N HCl or alkalifed with 1N NaOH and then boiled as above(pH was brought back to normal before testing) . The test material, dissolved in 0.05 M phosphate buffer, was incubated for 20 min with trypsin (0.1 mg/ml, pH 7.2), chymotrypsin (4 mg/ml, pH 7.4) and lipase (10 mg/ml) or 0.9%NaCl (controls) . Reaction was terminated by boiling. After appropriate volume adjustment, contractile activities of undigested (control) and enzymedigested material were compared . Isolated smooth muscle preparations For guinea-pig ileum, the terminal 2 cm strip was suspended in Tyrode at 37°C . Rat fundal strip was prepared by dissection according to the method of VANE (1957) and suspended in Tyrode at 37°C . For isolated rat uterus, a 2-3 cm section of the uterine horn was removed from a virgin albino rat (100-105 g) which had been brought into artificial oestrus by subcutaneous injection of 25 Ng/100 g body weight of stilboestrol suspended in arachis oil, 20-24 hr prior to the experiment . The uterine horn was suspended in de Jalon's solution at 28-29°C . Rat duodenum was suspended in de Jalon's solution at 28 -29°C . Hamster stomach strip was prepared according to the method of UaATUBA(1973) . All solutions were aerated with a mixture of 95% O, and 561o CO,. Contractions were recorded by a frontal writing lever on a moving Kymograph. To obtain contractions, preparations were generally exposed to agonists at the following concentrations (g/ml bath fluid) : acetylcholine (5 x 10-°); histamine (2 x 10-°) ; prostaglandin E, (1 .33 x 10-4 ); E, (1 .33 x 10-') ; F~ (8 x 10-') . However, if necessary, the concentrations were raised or lowered. For blocking agonists, the

355

Smooth Muscle Contractile Material

following were used (g/ml) : atropine (10-'), pretreatment for 120 sec; mepyramine, (10-'), pretreatment for 75 sec; methysergide (2 x 10-'), pretreatment for 30 min; polyphloretin phospate (2 x 10-'), pretreatment for 10 min . Antagonists under such conditions blocked the effects of ACh, histamine and serotonin on the smooth muscle preparations . Antagonist-treated tissues were exposed to agonists to ensure that contractions had been abolished. If this had not been achieved, antagonist concentrations were increased until contractions were abolished. Drugs used

Bradykinin (Sandoz, Switzerland), cyproheptadine (Merck Sharp and Dohme, India), polyphloretin phosphate (Leo, Sweden), prostaglandins E E, and F,. (Upjohn, USA) were obtained as kind gifts of the manufacturers. Acetylcholine chloride (E . Merck, India), atropine sulphate (Boehringer, W. Germany), chymotrypsin (3,xlcrystallised) (Sigma, USA), histamine acid phosphate (BDH, India), iodine (Sarabhai M. Chemical, India), indanetrione hydrate (BDH, India), mepyramine maleate (May and Baker, India), molybdenum oxide (BDH, India), 2-methoxy ethanol (BDH, India), potassium ferricyanide (E . Merck, India), Sephadex G-25 (Pharmacia, Sweden), 5-hydroxytyptamine-creatinine sulphate (E. Merck, India), Silica gel G type 60 (E . Merck, Darmstadt) and trypsin (2 x crystallised) (Sigma, USA) were obtained commercially . Solvents and chemicals, unless otherwise mentioned, were of analytical quality. Drug solutions were made up freshly. The drugs were prepared in 0.9% w/v NaCl, Tyrode solution or appropriate solvents as necessary. Concentrations are expressed for the free acid or base unless otherwise stated . In view of the difficulty of weighing the very small amount of active material present in individual gel filtrate aliquots, isolated tissue bath concentration of gel filtrate aliquot was expressed as volume per volume (v/v), i.e . volume of gel filtrate per volume bath fluid. RESULTS

Solvent extraction

The extraction of smooth muscle contractile activity with various solvents, expressed as percentage contractile activity (where pre-extraction material was 100%) were (mean of 3 experiments) : methanol, 71 .2% ; 95% ethanol, 50 .2% ; butanol, 50 .5% ; acetone, 0.75%; ethyl acetate, 0.04% ; ether, 0% .

Gel filtration

Crude venom was subjected to chromatography on Sephadex G-25 and the eluates were measured for their smooth muscle contractile activity on guinea-pig ileum and rat fundus . Eluate protein content was also measured (Fig. 1) . Gel filtered material (2.9 x 10-s , v/v) 0-5 00 3i

0-4 0. 3

75

É Oß O 0N

50

Z O F V Q C F Z O u

25 O TUBE

NUMBER

FIG . 1 . GEL FILTRATION ELUTION PROFILE OF CRUDE VENOM ON SEPHADEx G-25 . Fractions were collected with 0.05 M phosphate buffer, pH 7.2, at 4°C. Smooth muscle contractile activity of eluate [concentration (v/v) 2.9 x 10-°l was examined on isolated guinea-pig ileum pretreated with blocking doses of atropine - mepyramine - methysergide, against acetylcholine, histamine and 5-hydroxytryptamine, respectively . Absorption was determined at 280 nm for proteincontent. Contraction height was measured in millimeters.

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produced a quick contraction of guinea-pig ileum, starting almost immediately and reached a peak generally within 15 sec (Fig. 2). Gel filtered material (5 .8 x 10-4 , v/v) also contracted fundal strips, though sensitivity was less. The contraction generally reached a peak within 4 min after a latent period of 5 -10 sec (Fig. 2). Hamster stomach strip was also not very sensitive. Contractions were obtained with a concentration of 5.8 x 10-' (v/v) (Fig. 2).

j uu ~_J a oo 0

w a A à RF

FIG . 2. THE ACTION OF GEL FILTERED MATERIAL ON ATROPINE -MEPYRAMINE-METHYSERGIDETREATED ISOLATED GUINEA-PIG ILEUM, HAMSTER STOMACH AND RAT FUNDAL STRIP .

Time-scale in min . GPI, guinea-pig ileum ; " , gel filtered material, 2 x 10- " (v/v); O, gel filtered material, 2 .9 x 10- ' (v/v) . HSS, hamster stomach strip ; A, gel filtered material, 2 x 10-1 (v/v); A, gel filtered material, 5 .8 x 10-4 (v/v) . RF, rat fundal strip ; " , gel filtered material, 5 .8 x 1V (v/v) .

On rat duodenum, gel filtered material (1 .4 x 10-3, v/v) produced a contractile response, in contrast to bradykinin which produced relaxation (Fig. 3). Gel filtered material (3 x 10-°, v/v) produced a slow contraction in the rat uterus . After a lag period of 10-15 sec it reached a peak in about 45 sec (Fig. 3) . In a series of ten experiments for each of the isolated tissue preparations mentioned above, responses were reproducible ; there was no tachyphylaxis.

FIG 3 . THE ACTION OF GEL FILTERED MATERIAL AND BRADYKININ ON ISOLATED RAT UTERUS AND DUODENUM . Time-scale in min . RU, rat uterus ; O, gel filtered material, 3 x 10' (v/v) . RD, rat duodenum ; [1, gel filtered material, 1 .42 x 10-' (v/v) ; O, bradykinin, 2 x 1V g/ml .

Thin layer chromatography A spot (RF 0.6) was obtained using solvent system A when crude venom was applied on

35 7

Smooth Muscle Contractile Material

TLC. This spot was distinct from spots produced by authentic ACh (RF 0), histamine (RF 0), 5-HT (RF0) and prostaglandin (R, 0). When material obtained by Folch extraction was subjected to chromatography with solvent system A, and developed either with iodine vapour, ninhydrin or molybdenum reagent, a single spot (R F0.65) was found. When venom was subjected to TLC with solvent system B and developed similarly, three spots were found (R F 0.1, 0.2 and 0.3). In all cases, parallel spots were scraped off, extracted, vacuum-dried, reconstituted with physiological solution for testing spasmogenicity on isolated atropine - mepyramine - cyproheptadinetreated guinea-pig ileum. An arbitrary unit was chosen to express smooth muscle contractile activity quantitatively . The activity of 40 ng of a pooled sample of crude venom on guinea-pig ileum pretreated with atropine - mepyramine - methysergide was designated as one unit. Material at various stages of purification was assayed against this in terms of contraction of atropine - mepyramine - methysergide-treated guinea-pig ileum, and expressed in terms of units of activity . Measured in terms of units/mg phosphorus, purification achieved was as follows: gel filtration, 3 .1-fold; thin-layer chromatography, 13.3-fold. Biochemical nature of smooth muscle contractile material The protein peak can be clearly distinguished from the contractile peak, and no protein could be shown by the Lowry method in the eluate which caused contraction (tubes 9 -15, Fig. 1) . Inorganic phosphorus, amino nitrogen and amino sugar content in the various fractions are shown in Table 1 . The anthrone test was positive, but when the hydrolysis step was omitted from this test no sugar could be detected . TABLE 1 . AMINOPHOSPHORUS, NITROGEN AND SUGAR CONTENT IN FRACTIONSOF VENOM

Fractions Venom (I)" Gel filtrate material from I (=II)' Solvent extracted material from II (= III)' TLC extracted material from III (=IV)' Fractions from IV : SL I t SL2 t SL3 t

Amino phosphorus 203 t 3

Amino nitrogen 137 t 3

49

t5

121

t3

100

t1

32

t1

50

t1

69

t1

22

tl

15 .5 t 0.6

30 .2 t 0 .5

Amino sugar

10.0 t 0.8

76 t 2 5.3 t 0.2 13 .9 t 1 .3 191 t 4 47 .6 t 1 .4 24 t 1.8 236 t 9 14 .1 t 1 .2 52 .5 t 4.8 All values are mean t S.E . (N=4) 'Expressed in terms of Mg/mg of the fraction under study. t Expressed as 1Ag/mg of TLC extracted material .

Following the procedures described under Materials and Methods, loss of contractile activity occurred with heat-treatment (30.4%), acid-treatment (50%B) and alkali-treatment (73%) . Digestion with trypsin, chymotrypsin or lipase did not result in any loss of contractile activity . Experiments to distinguish smooth muscle contractile material from prostaglandins Since solubility and separation characteristics suggested a lipid nature for smooth muscle contractile material, attempts were made to distinguish it and its components from

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prostaglandins by use of blockers, differing TLC mobility and differing adsorption chromatography elution profiles, and estimation of Gaddum's discrimination index by parallel bioassay . Gaddum's discrimination index is based on the fact that whatever the test system, bioassay of a given amount of an agonist in terms of the same agonist would yield the same or very nearly the same value in every case, so that the highest value divided by the lowest value (discrimination index) should be nearly 1 . However, if an agonist is bioassayed in terms of a different agonist on several test systems, the values will differ according to the varying sensitivities of the tissues to the two agonists, so that discrimination index values are likely to be higher than 1 . e

n

I_j LJ L-J L __J.oVzJ . . a o

a_-4_--0----0

J e

J L--_j a e

o-__a-__o. -- -'o

FIG. 4. EFFECT OF POLYPHLORETIN PHOSPHATE ON THE CONTRACTILE EFFECT OF SMOOTH MUSCLE CONTRACTILEMATERIAL IN GUINEA-PIG ILEUM ANDRATFUNDAL STRIP. Time-scale in min. Top panel - guinea-pig ileum. ", Smooth muscle contractile material, 2 x 10-" g/ml ; A, prostaglandin E 1 .33 x 10-4 g/ml ; A, prostaglandin E,, 1.33 x 10~ g/ml ; O, polyphloretin phosphate,2 x 10 -. g/ml . Bottom panel - rat fundal strip. A, Prostaglandin F,., 8 x 101 g/ml ; ", smooth muscle contractile material, 2 x 10-° g/ml ; O, polyphloretin phosphate, 4 x 10 4 g/ml .

Polyphloretin phosphate abolished the action of PGE, and PGE, on guinea-pig ileum and PGF,, on rat fundal strip, but not that of smooth muscle contractile material on either of these preparations (Fig. 4). A parallel bioassay was carried out for smooth muscle contractile material in terms of PGE,, PGE, and PGF,, on guinea-pig ileum, rabbit jejunum, rat fundal strip and hamster stomach. The discrimination index was far greater than the value of 1, which would be expected were the unknown agonist identical with the known agonist (Table 2). On thin layer chromatography (chloroform - methanol - water), the R, values of the smooth muscle contractile material was 0.65, while corresponding values for prostaglandins E,, E, and F,, were 0, 0 and 0.1, respectively . With chloroform - methanol - acetic acid - water, the R, value of smooth muscle contractile material was 0, and those of prostaglandins E,, E, and F,, were 0.88, 0.78 and 0.69, respectively . On adsorption chromatography, the elution characteristics of prostaglandins E, and F,, varied markedly from that of smooth muscle contractile material . Using a methanol - ethyl acetate -toluene eluting system, E, was eluted with a mixture of 0.7 : 64 .3 : 35, respectively ; F,, with 1 : 79 : 20; smooth muscle contractile material with 100 : 0 : 0. With methanol - chloroform - ether, prostaglandins E, and F,, could not be eluted, but smooth

Smooth Muscle Contractile Material

35 9

TABLE 2. PARALLEL BIOASSAY OF SMOOTH MUSCLE CONTRACTILE MATERIAL ON DIFFERENT ISOLATED SMOOTH MUSCLE CONTRACTIONS IN TERMS OF PROSTAGLANDINS E,, F, AND F,,

Preparations* Gaddum DiscriminGuinea-pig Rat Rabbit Hamster ileum fundus gut stomach ation index Prostaglandin E, t 0.024 0.03 29 1160 Prostaglandin F,fi 48 0.14 0.06 0 .005 9600 Prostaglandin F, .t 16 0.34 0.06 0.006 2666 *Each entry represents the mean of five experiments . tAmount of prostaglandin (Mg) equipotent with 1pg of smooth muscle contractile material . Agonist against which assayed

muscle contractile material started to appear at 90 : 0 : 10, and was completely eluted in the 100 : 0 : 0 fraction . DISCUSSION

Earlier work by Nag Choudhuri (Ph.D . Thesis, 1976) had shown that crude venom of Heterometrus bengalensis produced smooth muscle contraction of several isolated preparations . These contractions could not be blocked by antagonists of ACh, histamine or S-HT . This suggested the presence of a substance(s) other than those mentioned above. Solvent extraction could partly separate smooth muscle contractile material . The solubility pattern found with single solvents suggested the possibility that the smooth muscle contractile material was a lipid, because of its high solubility in lipid solvents . It is not likely to be a protein in view of the negative Lowry test and lack of optical density at 280 mm and because gel filtration yielded no protein in the contractile peak, the protein peak being distinctly separate . The smooth muscle contractile material in Heterometrus bengalensis venom seems to be a mixture of three hitherto-unknown substances. For convenient reference, and pending full chemical identification, the present workers refer to the mixture as substance L (L for lipid), the three components being designated provisionally as substances L1, L2 and L3. TLC of crude venom with solvent system A showed a single spot, the eluate of which (substance L) contracted atropine - mepyramine - cyproheptadine-treated smooth muscle . However, resolution of this spot with solvent system B indicated that Substance L was comprised of at least three components, SL1, SL2 and SL3, each with contractile activity . The presence of a smooth muscle contractile lipid component in scorpion venom seems exceptional. No such lipid had been reported from the venom of other species of scorpion . Some scorpion venoms do contain lipid, however. L . quinquestriatus venom has a lipid content of 1 .7% (phosphatidyl ethanolamine, phosphatidyl serine, lecithin and sphingomyelin) but no biological activity has been reported as being due to these lipids (MARIE and IBRAHIM, 1976). KABARA and FISHER (1969) reported the presence of extractable lipids in Naja naja venom which were not lethal to mice, though they reported that 40.8% of the lethality of the venom was lost on extracting the lipids . Turning to fish, HATANO (1974) has reported the presence of a toxic lipid containing adenine (dinogunellin) in the roe of the northern blenny, which induced gastrointestinal disorders. In view of the solubility, separation characteristics, TLC mobility and the presence of phosphorus, one would think that substance L was a phospholipid . The material contains amino nitrogen, the amount decreasing with progressive purification . Amino nitrogen along with inorganic orthophosphorus in Substance L and its fractions suggests that they

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are nitrogenous phospholipids, which, as expected, could be developed on TLC with iodine vapour, ninhydrin or molybdenum reagent. Substance L could be distinguished from prostaglandins E,, E, and F,, by differences in response to a prostaglandin blacker, TLC mobility, adsorption chromatography elution profile and the discrimination index of Gaddum. Substance L gave a positive reaction with Molisch's test but was negative with Benedict's test. The standard anthrone test was positive, but it was negative when the hydrolysis step was omitted; probably there was a nonreducing sugar moiety/moieties freed by hydrolysis . SL3 contained the highest amount of sugar . The presence of sugar moiety/moieties in a spasmogenic component in scorpion venom has not been reported earlier. The present data do not permit firm conclusions regarding the structure of SL and its components, however, one may perhaps speculate about a phosphatidyl glycerol with a sugar moiety or moieties attached, possibly to the base of the phosphatidyl residue in the a-position. Acknowledgements - This work was supported partly by the Indian Council of Medical Research and partly by

a Postgraduate Grant in Forensic Sciences, Bureau of Police Research and Development. Ministry of Home Affairs, New Delhi. The authors express their thanks to Dr. JYGTI DATTA, Reader in Chemistry, Bose Institute, Calcutta, for his valuable advice and to Dr . JOHN E. PIKE of Upjohn Company for the generous gift of prostaglandins .

REFERENCES BARTLETT, G. R. (1959) Phosphorus assayin column chromatography, J. biol. Chem. 234,466 . BLIGH, E. G. and DYER, W. J. (1959) A rapid method of total lipid extraction and purification. Can . J. Biochem. Physiol37, 911 . CuNHA-MELo, J. R., FREIRE-MAIA, L., TAFURI, W. L. and MARIA, T. A. (1973) Mechanism of action of purified scorpion toxin on the isolated rat intestine. Toxicon 11, 81 . DEL Pozo, E. D. (1956) Mechanism of pharmacological actions of scorpion venoms, In : Venoms, p . 123 (BUCKLEY, E. E. and PORoEs, N., Eds.) . Washington : American Association for the Advancement of Science. DINIz, C. R. and GoNcALvEs, J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venom, In : Venoms, p . 131 (BUCKLEY, E. E. and PORGES, N., Eds.) . Washington : American Association for the Advancement of Science. Direz, C. R. and VALERI, V. (1959) Effect of a toxin present in a purified extract of telsons from the scorpion Tityusserrulatm on smooth muscle preparationand in mice . Archs Int . Pharmacodyn. Thér. 121, 1. DiNiz, C. R. (1971) Chemical and pharmacological properties of 77tyus venoms . In : Venomous Animals and their Venom, Vol . III Venomous Invertebrates, p . 311. (BucHERL, W. and BUCKLEY, E. E., Eds.) . New York : Academic Press. Fists C. H. and SuBBAROw, Y. (1925) The calorimetric determination of phosphorus . J. biol. Chem . 66, 375. FoLcH, J., LEES, M. and SLoANE STANLEY, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226, 497. GADDum, J. H. (1955) Polypeptide which stimulates plain muscle, In : Index of Discrimination, pp . 133 -135 (GADDum, J. H., Ed .) . Edinburgh: E. & S. Livingstone. HATANo, M. (1974) Properties of a toxic phospholipid in the northern blenny roe . Taxiton 12, 231. ISMAIL, M., OSMAN, O. H. and EL-AsmAR, M. F. (1973) Pharmacological studies of the venom from the scorpion Buthus minax (L. Koch). Tadeon 11, 15 . ISMAIL, M., OSMAN, O. H., Gum" K. A. and KARRAR, M. A. (1974) Some pharmacological studies with scorpion (Pandinus exidalis) venom. Taxiton 12, 75 . KABARA, J . and FISHER, G. H. (1969) Chemical composition of Naja naja extractable lipids . Toxicon 7, 223. LowRY, O. H., RGBEBRGUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin-phenol reagent, J. blol. Chem. 193, 265. MARIE, Z. A. and IBRAHRN, S. A. (1976) Lipid content of scorpion (Leiurus quinquestriatus, H. & E.) venom. Toxicon 14, 93 . RosEN, H. (1957) A modified ninhydrin calorimetric analysis for amino acids. Archs Biochem. Biophys. 67, 10. SERGENT, E. (1947) Scorpion antivenae.Bull. Acad. natn., Mid. Paris 131, 45 . STAHL, E. (1965) The application of thin layers to carrier plates . In : Thin Layer Chromatography. (STAHL, E., Ed .). New York : Academic Press.

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F. (1968) Pharmacological properties of the toxins of scorpion venom (Androctonus australis) . C. r. hebd. Séanc. Acad. Sci., Paris26713, 240. TAZIEFF-DEPIERRE, F. (1972) Venin de scorpion, calcium et emission d'acetylcholine par les fibres nerveuses Fileon de cobaye . C. r. hebd. Séanc. Acad. Sci., Paris27513, 3021 . TREVELYAN, W. E. and HARRISON J. S. (1951) Studies on yeast metabolism . Fractionation and microdetermination of cell carbohydrates. Biochem. J. 50, 298. UBATUBA, F. B. (1973) The use of the hamster stomach in vitro as an assay preparation for prostaglandin. Br. J. Pharmac. 49,662 . VANE, J. R. (1957) A sensitive method for the assay of 5-hydroxytryptamine . Br. J. Pharmac. 12, 344. WHITTERMORE, F. H., JR ., KEEGAN H. L ., FITZGERALD, G. M., BRYANT, H. A. and FLANIGAN, J. F . (1963) Studies on scorpion antivenins, venom collection and colony maintenance, Bull. WidHith Org. 28, 505 . TAZIEFF-DEPIERRE,