Stonustoxin-induced vasorelaxation is inhibited by the specific nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester

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Crystals of a platelet aggregation inhibitor, the acidic PLA efrom the venom ofAgkistrodon halys pallas. L. GuI, X. NIU, J. YANG, R. Bi, Z. LIN and Y. CimN (Institute of Biophysics, Academia Sinica, Beijing 100080, P.R. China). Tim ACIDIC phospholipase A 2 from Agkistrodon halys pallas consisting of 124 residues belongs to a class of phospholipase A2 which inhibits platelet aggregation. Structural studies of this PLA2 and homologous neutral PLA2, with presynaptic toxicity from the same venom, will increase our knowledge of understanding the structural basis of the function of this protein. The acidic PLA 2 was purified by previously described techniques (Acta Biochem. Biophys. Sinica 16, 664, 1984) and crystallized by vapour diffusion method. The crystals were obtained from aliquots, containing 2, 5-hexandiol, 0.1 mg/l Na2ASO3-HCI and 15 mg/ml protein, which were equilibrated against 65% 2, 5-hexandiol solution. The crystals grow as long hexagonal prisms and diffract to a resolution better than 2.5 A. Some crystal data were determined by a CAD-4 diffractometer. The space group is P6 t with cell dimensions a = b = 83.2/~ and c = 32.6/k. There is only one molecule per asymmetric unit. Thus the crystals are excellent candidates for a successful X-ray analysis. Diffraction data have been collected to 2.5 A resolution using an area detector. The structural analysis by molecular replacement methods based on the presumed structure homology between this PLA 2 and crotalid PLA 2 is underway.

Yield and molecular weight of the Duvernoy's gland secretions of the dog-faced mud snake Cerberus rynchops (Serpentes: Colubridae; Homalopsinae). M. L. GUINEA, L. MCMORROW and N. PEERZADA(Faculty of Science, Northern Territory University, P.O. Box 40146, Casuarina 0811, Northern Territory, Australia). DUVERNOY'Sgland secretions of Cerberus rynchops were tested to determine the general toxicity and the mol. wt and concentration of the protein component. Secretions were collected by aspiration from the fang surface. Yields were in the order of 10/al per snake. Protein tool. wts were determined using isoelectric focusing with polyacrylamide gel electrophoresis. Major bands indicated the presence of proteins with mol. wts 41,000~6,000, 28,000, 27,000 and 16,000 with minor bands of proteins with those of 73,000, 61,000-64,000, 53,000, 20,000-21,000 and 14,000. Clotting times were increased by 12% using activated partial thromboplastin time. Comparisons are made where possible with secretions from the Duvernoy's gland of Enhydris polylepis. This initial investigation into the toxicity of Australian homalopsins encourages further investigations.

Stonustoxin-induced vasorelaxation is inhibited by the specific nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester. M. C. E. GWEE,l K. S. Y. Low, I R. YUEN,2 H. E. KHOO2 and P. GOPALAKRISHNAKONE3 (Departments of JPharmacology, 2Biochemistry and 3Anatomy, Faculty of Medicine, National University of Singapore, Lower Kent Ridge, Singapore 051 I). STONUSTOXIN, a purified peptide toxin from the venom of the stonefish, Synanceja horrida, can cause an endothelium-dependent relaxation of the rat aorta precontracted with noradrenaline. The present study investigates the possible mechanism(s) by which stonustoxin produces the endothelium-dependent relaxation. The descending aorta from adult Sprague-Dawley rats was removed, cut into helical strips and mounted under l-g tension in Krebs solution gassed with 5% COJ95% O 2. Responses were measured with a Ugo Basile Model 7006 isotonic force displacement transducer. The nitric oxide synthase inhibitor N°-nitro L-arginine methyl ester (L-NAME) (25/aM) prevented stonustoxin-induced relaxation of the rat aorta previously precontracted with noradrenaline (25nM). However, L-arginine (75/aM), but not D-arginine (75/aM), reversed the inhibition produced by L-NAME. Relaxation produced by sodium nitroprusside was not inhibited by L-NAME. The results strongly suggest that relaxation of the precontracted rat aorta was mediated via the release of an endothelium-dependent relaxing factor which is most likely to be nitric oxide.

Chemistry and pharmacology of amphibian venoms. G. G. HABERMEHL(Department of Chemistry, Veterinary University, Bischofsholer Damm 15, D-3000 Hannover, F.R.G.). Tim SKIN of amphibians (toads, frogs, newts, salamanders) contains numerous small glands covering all parts of the body. They contain a wide variety of substances in chemical as well as in pharmacological respects. The main purpose of most of these compounds is the protection of the skin against infections by microorganisms. Many of them, however, are potent toxins, acting on different sites of an organism, among others as neurotoxins. o-Methyl-bufotenin, for example, acts as an hallucinogen, batrachotoxin opens the sodium channels irreversibly, while tetrodotoxin blocks these irreversibly. Samandarin acts on the CNS causing convulsions and paralysis. Others, as short chain peptides, act as hypotensive or hypertensive agents or as vasoconstrictors, and another group possesses nerve-muscle activity, probably affecting the calcium channels. Apart from these interesting

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activities, some of these compounds have become valuable tools in the studies of channels or receptors. In this respect, the structure/activity relationship is of great importance. In some cases, by chemical synthesis, compounds have either been changed marginally or have been synthesized de novo with other stereochemistry. As far as we know, toxicity, as well as other properties, may be changed completely as well as only partly; to mention just one example: the optical antipode of pumiliotoxin-C is ten times less toxic in mice than the natural PTX-C, but still retains the whole activity against microorganisms. Quite recently, we have found low mol. wt compounds in snake venom closely related to some of the amphibian peptides, and with potentiating or similar activities. The same is true for some substances we could isolate from spider venom. It is interesting to see this parallel evolution. Obviously, certain structure types have turned out to be extremely successful.

Ciguatoxin-soluble protein complex from skeletal muscle of Scomberomorus commersoni. S. T. HAHN and M. F. CAPRA (Centre for Biological Population Management, Queensland University of Technology, Australia). A STUDYof soluble protein from the skeletal muscle of Scomberomorus commersoni was undertaken to elucidate aspects of CTX-bioaccumulation in marine teleosts. Skeletal muscle tissue samples from toxic and non-toxic specimens were subjected to fractionation, centrifugation, (NH4)2SO4 precipitation and Sephacyl S-200 chromatography for soluble proteins. Toxicity associated with various fractions was assessed by mouse bioassay. Toxic and non-toxic soluble protein fractions were compared visually using SDS-PAGE. CTX eluted from Sephacryl S-200 with soluble proteins between 35,500 and 59,500 mol. wt. The toxic eluate contained 1.4% of total sample protein and 15% of total sample toxicity, with an associated 7.2-fold increase in specific activity. SDS-PAGE comparisons show two protein bands between 37,400 and 40,600 mol. wt, unique to toxic soluble protein fractions. A CTX-protein complex composed of at least one monomeric protein between 37,400 and 40,600 mol. wt was identified in the soluble protein derived from S. commersoni skeletal muscle.

Presentation of a haptene influences antibody specificity: an example with a peptide selected in the cholera toxin B sub-unit. H. HALIMI, G. MILHAUDand P. RIVAILLE(LA 163 CNRS CHU St. Antoine 27 Rue Chaligny, 75012, Paris, France). THE SEQUENCEP-50-75 selected in the cholera toxin B sub-unit was orally or intraperitoneally administered to C 57 BI mice without carrier and adjuvant in three forms: alone; as an octamer synthesized on the 'octopus' proposed by Tam in 1988 (S); or on the e-NH 2 a chain of eight lysines, each one spaced by two glycines (C). Whatever the mode of presentation to the immune system, the titres of seric antibodies against the cholera toxin were not especially increased. But mice orally immunized with (C) were the most protected against the toxin. In contrast to P 50-75 and in a lesser degree of the B sub-unit, (S) and (C) are able to increase, at the intestinal level, the amounts of secretory IgAs, total or specific to the toxin, these IgAs being able to neutralize the toxin activity. Amounts of different isotypes recognizing the toxin (IgM, IgG1, IgG2a, IgG2b, IgG3) depend on how the antigen P 50-75 is presented to the mice when mice are primed with peptides and boostered with B sub-unit.

Dendrotoxin homologues block voltage-dependent potassium currents in cultured rat dorsal root ganglion cells by different mechanisms. A. HALL, J. STOW, O. DOLLY and D. OWEN (Department of Biochemistry, Imperial College, London and Wyeth Research (U.K.), Huntercombe Lane South, Taplow, Berks, SL6 0PH, U.K.). THE SNAKEvenom toxins, dendrotoxin (DTx) and Toxin I, are potent blockers of voltage-activated K + currents in sensory neurones. A number of homologues of DTx and Toxin I have been identified in the crude venom of green and black mamba snakes, respectively. We have determined, electrophysiologically, the K+channel blocking activities of additional members of the dendrotoxin family. Dorsal root ganglion cells (DRGs) were collected from newborn rats and grown in culture for 3 to 8 days before recording. Under these conditions the cells expressed two major components of voltage-activated K ÷ current; slowly-inactivating (z = c. 50msec, 200 msec and 2sec) and non-inactivating ( > 2 min). All homologues tested, i.e. ,t-, fll r, f12-, ~-, 6-DTx, Toxin I and Toxin K, blocked outward current activated from-100mV at concentrations between 10nM and 1 #M. In particular, the ECs0s for c¢- and 6-DTx were c. I nM and 0.5 nM, respectively, with maximal block of 25-50% total outward current. Qualitative differences in the block were noted between homologues, e.g. whereas ct-DTx preferentially blocked the early phase of currents activated by a depolarizing voltage step, 6-DTx appeared to block in a time-dependent manner, leaving the peak current relatively unscathed. Qualitative differences were also seen for other homologues. Analysis of difference currents showed an apparent selectivity for different components of outward current, ct-DTx blocking an inactivating component and 6-DTx a non-inactivating component, ct-DTx did not alter the half maximal activation potential ( + 5 mV). Tail current analyses revealed two time constants of deactivation (c. 5 msec and 40-90 msec at -- 80 mV) which appeared to correspond to the inactivating and non-inactivating components, respectively, since the fast component is reduced at both