Activity of cytotoxin P4 from the venom of Naja nigricollis after various chemical modifications

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8th European Symposium-Abstracts

Addition of CIII or EQII to one side of a planar lipid membrane (HLM) increase the conductivity of the film in discrete steps of defined amplitude. High pH and sphingomyelin promote the interaction even in this system . We suggest that sea anemone cytolysins increase membrane permeability by forming discrete ion channels in it . REFERENCES Bextatn:n~te, A. W. and Rvnv, B. (1988) Biochim. Biophys. Acta 884, 12~141 . MENE3rRINA, G. (1988) FEBS Lett. 232, 217-220. Effects of myotoxin a on cultured muscle cells. ALLAN L. H®ea, H~~a A. Mrrnctnsox and C~r~ter.rs H. 7ror .tcowstn (Department of Chemistry, Arizona State University, Tempe, AZ, U.S .A.) . MYOTOXIN a is a small basic protein, isolated from the venom of Crotalus viridis viridis that causes extensive damage to muscle tissue when injected . We have used cultured myoblasts and the myotubes that result from cell fusion to study the effects of purified myotoxin a and some chemically modified forms of the protein . Standard cell culture techniques were used to establish and maintain clonal cell lines and primary cultures derived from neonatal rat skeletal muscle. Toxins were added to the cultures at concentrations which ranged from 0.0~ 1 .0 pM under well-defined culture conditions . The presence of myotoxin did not appear to affect the fusion process of primary myoblast cultures . Similar experiments with some clonal cell lines, however, led to the formation of very large myotubes compared to the untreated control cultures. Myotubes derived from primary cell cultures twitched spontaneously, but the twitching stopped when the normal growth medium was replaced with a defined serum-free medium . Addition of myotoxin a to the myotubes in the serum-free condition caused the twitching of the myotubes to resume . Crotamine, a closely related myotoxin from C. d. terriftcrrs venom, gave comparable results. Products derived from reductive alkylation, nitration and alkylation of myotoxin a were used in analogous experiments and were nearly devoid of activity when tested with the serum-free cell culture system . The results demonstrate the utility of muscle cell cultures in studies of myotoxin a and related compounds. Supported in part by National Institutes of Health; # 5 RO1 GM34925. Structure-junction relationships of tetanus toxin. HERNARD Brzztrtr, (Pasteur Institute, Paris, France). TsrAxus toxin has been extensively studied over the last two decades. The physicochemical and toxicological characteristics, as well as the secondary structure of the toxin purified to homogeneity have been specified. The toxin molecule has been sequenced. The binding characteristics have been determined and the axoplasmic and transynaptic movement of the toxin demonstrated. However, its mode of action is still to be clarified. In this lecture we shall report on our own contribution to the studies on tetanus toxin in relation to the structur~function relationships: mechanism of tetanus toxin detoxification by formaldehyde action and its implication for vaccine preparation; involvement of particular amino acid residues in toxin action; toxicity and immunological reactivity and significance for the preparation of therapeutic antisera; protease cleavage of the toxin molecule, purification from the cleaved molecule of a fragment ; H~, involved in binding and transport of the toxin molecule to the CNS and of another fragment, L-H,, responsible for the paralytic (botulinum toxin-like effect); use of the HZ fragment for targeting an enzyme or a drug to the CNS; application to the treatment of a grafted brain tumour ; use of HZ as a tool for investigating the bases of "neurotropism" ; its interaction with the fate of a neurotropic virus inoculated by i.v. or i.m. routes; investigation of the pathogenic action of fragment L-H, in its native state, in situ in the toxin molecule; interaction of the toxin molecule and the L-H,-derived fragment with guanylate cyclase. Activity of ~yotoxin P, from the venom of Naja nigricollis after various chemical modt~cations. GAnt HORKOW, AnrxA C~rArur-MA1YA3 and MrcxAaL t)vAnrA, (Department of Zoology, Tel Aviv University, Israel 69978). Ttm vsxoes of Naja nigricollis includes four cytotoxic factors (P 3P~, Ctwta-MA~rvAS and twAnu, 1989). The myotoxin P, was isolated in three steps: (l) reduction by 1% 2-mercaptcethanol; (2) gel filtration on Sephadex G-50; (3) ion-exchange chromatography on CM-Sepharose. The cytotoxic activity was tested on melanoma cells B16F10 and on WEHI-3B leukemia cells. The purified myotoxin P, was modified by various reagents reacting with different residues of the protein. The N-terminal and the C-terminal groups of the polypeptide were modified by N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or cystamine respectively; these reagents include 5-S bonds. Both modified polypeptides retained about 90% of their original cytotoxic activity . Moreover, reduction of the S~ bonds of the bound reagents in mild conditions exposed the SH groups on the reagents without affecting the cytotoxicity of the polypeptide . However, at extreme reducing conditions, the integral S-S bonds of the ~yotoxin were also reduced, leading to a total loss of the cytotoxic activity . Similarly, other modifications such as succination, maleylation, reductive alkylation, carbamylation, reaction with 1,2cyclohexanedione or complete iodination of the toxin also abolished the cytotoxic activity, but did not affect its

8th European Symposium-Abstracts

14 5

capability to bind to cells . The reaction of the SPDP or cystamine with the N-terminal or C-terminal residues has special importance as these reagents do not affect the cytotoxic activity significantly and, therefore, may be used to conjugate the cytotoxin with another molecule such as specific monoclonal antibodies generated against malignant cells . REFERENCE GüAIM-MATYA3, A . and Ov~wre, M . (1989). Isolation of a cytotoxic factor from the venom of Naja nigricollis preferentially active on melanoma cells. Toxicon 27, 35 . On the inhibition of [Na+,K+]-ATPases by phospholipases A Z. Pm~tnE E. Houors, Arcu.~ Krna .~ and Hertv~ ROCHAT (CNRS UA1179-INSERM U172, Laboratoire de Biochimie, Faculté de Médecine Nord, Bd P . Dramard, 13326 Marseille Cédex 15, France). Try vexoM of Naja mossambèca mossambica contains three toxic phospholipases AZ (PLA Z) enzymes as well as several non-PLAZ cytotoxins called cardiotoxins . Previous studies produced data suggesting that the cardiotoxins inactivated [Na',K+]-ATPase (ATPase) and this action may play a role in the toxicity of the venom. We have compared the effects of purified PLAIS and cardiotoxins from Naja m. m. venom on the catalytic activities of ATPase and [K+]-PNPase (PNPase) from rat brain . Our results were the following: (1) micromolar concentrations of cardiotoxins were required to inhibit ATPase and PNPase activities to the extent achieved by picomolar concentrations of the PLAI S; (2) Comparing PLAzs from diverse sources, a correlation was observed between PNPase inhibition, isoelectric point and toxicity for mice; (3) Using rat brain membranes, extended incubation times with the most basic Naja m . m . PLAZ presented a biphasic PNPase inhibition curve, suggesting that two PNPase activities were affected differently . In contrast, treatment of rat brain membranes with either porcine pancreatic PLAZ or beta-bungarotoxin or erythrocyte membranes with Naja m . m . PLA Z produced monophasic PNPase inhibition curves . In total, our results raise the possibility that the apparent ATPase inhibitory activities of cardiotoxins may be due to contaminating PLAS . Furthermore, the effects of PLA IS on ATPases are complex and a function of both the source of PLA Z and target membranes. We discuss a possible specific action of toxic, basic PLAZ on one of the [Na+,K']-ATPase isoforms of excitable membranes . Mamma!- and insect-directed .scorpion rreurotoxins: cloning and expression . P~xxE E . BOUGr3,' H . RoeFUr' and LEONARD A . SMrrtrZ ('Université d'Aix-Marseille II, Faculté de Médecine-Nord, Biochimie, UDC: CNRS UA 1179-INSERM U 172, Bd P. Dramard, 13326 Marseille, Cedex I5, France, and rfoxinology Dept ., Pathology Div., USAMRIID, Fort Detrick, Frederick, MD 2(701, U .S .A .). A3 PART Of our continued interest on the immunochemistry and the structure-activity relationships of scorpion neurotoxins, we have undertaken the molecular cloning and the expression of these toxins . We isolated fulllength cDNAs encoding Androctonus australis scorpion neurotoxins active on mammals or on insects . Sequence analysis of I1 cDNAs revealed that precursors of toxins contained signal peptides of about 20 amino acid residues . In addition, precursors of toxins active on mammals had diverse peptide extensions at their C-terminals. Accordingly, the processing steps required for maturation of precursors into toxins were not identical for all toxins. Southern blot analysis performed at the genomic level with toxin II cDNA suggested a single copy gene . Finally, as the first successful attempt to express animal toxins, we demonstrated that monkey kidney COS-7 cells transfected with a plasmid harboring toxin II cDNA transiently expressed a biologically active recombinant toxin. Effects of equinatoxin !I on the isolated guineapig heart . M . BUDIHNA,' P. MAL'EK2 and D . $urur° ('Institute of Pharmacology, Faculty of Medicine, ZDept. of Biology, Biotechnical Faculty, 3 Institute of Pathophysiology, Faculty of Medicine, E.K . University, Ljubljana, Yugoslavia) . PHARMACOIAGtCAL effects of Equinatoxin (EqT), a lethal protein isolated from the sea anemone Actinic equina L . (FERLAN and LEaez, 1974), have been studied on whole animal (Srcgr et al., 1974 as well as o~n, guinea-pig heart (L,~ et al., 1983), atrium (Ho et al., 1987) and on various excitable membranes (~upur, 1986; JUPUT et al., 1987; Surur, 1988) . Lethality caused by the toxin was assumed to be related to the cardiorespiratory failure (Sket et al., 1974) or cardiotoxicity alone (Ho et al., 1987; ~`urur, 1986) . Recently three toxins (EqT I, II, and III) have been isolated from the Actinic equina (MAi`Erc and LEeEZ, 1988) . As r .n~ for EqT and EqT II are practically identical (app . 35 ug per kg in mice) it seemed reasonable to investigate effects of EqT II on guinea-pig heart . For further characterization of the effects of EqT II the coronary flow, the heart rate, contractility of left ventricle, and ECG were recorded . Effects of EqT on these parameters are dose-dtpendent and show striking tachyphylaxis. Negative inotropic effect together with marked coronary vasospasm and abnormalities of ECG