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Cloning and nucleotide sequence of crotamine genes
838
2nd International Symposium
and Glu analyses using RP-HPLC with fluorometric detection after OPA procolumn derivatization .' The basal outflow of Asp/Glu in this preparation was 10-30pmole/min . A significant Ca 2`-dependent increase was observed after 3 min K" 20 mM application. When added to the superfusion medium, DTX significantly increased the release of ASP and GLU. After the initial increase, the amino acid levels gradually returned to basal values . The effect of DTX was partially Cap'-dependent and was not abolished by pretreatment with D-ASP. A dose-dependent effect was scen between 10_ 6 to 10 -9 M DTX. At 7 x 10 - b M, the DTX-evoked release was 300% for Glu and 160% for Asp respect to basal values. After 20 min of continuous DTX superfusion synaptosomes failed to further GLU release when depolarized with 20 mM K" in wntrast with a 600% increase in control microcolumns . These results suggest that DTX induces the release of a vesicular pool of Glu and, to a lesser extent, of Asp, leading to a depletion of the transmitter pool, suggesting that excitatory DTX effects could be related to an increased release of excitatory amino acids in the CNS . This work was partially supported by LP.LC.S. (International Program in Chemical Sciences, Uppsala, Sweden) . F LINDROTH, P. and MorrFrt, K. Analyt . Chem . Sl, 1667 (1979).
Some pharmacological aspects of iris acetylcholinesterase inhibition by jasciculins. R. Savatan, J. MAZZELIA, J. STrextvsct~t~rrtz and F. DN~s (Neurochemistry Div., Instituto de Investigaciones Biologicas Clemente Estable. Montevideo, Uruguay and Laboratory of Glaucoma Research, Pharmacia Ophthalmic AB, Uppsala, Sweden). the cholinergic innervation of the eye participates in several physiological functions, such as the regulation of papillary size, intraocular pressure, ocular circulation, etc., it appeared important to study the role of two peptides, called fasciculins (FAS), recently isolated from the venom of the green mamba snake Dendroaspis angusticeps which have been demonstrated to be powerful inhibitors of acetylcholinesterase (AChE) . Injected in the anterior chamber of rabbits' eyes, FAS (5-30 Ng, total dose) provoked a marked miosis and a significant inhibition (more than 50%) of iris AChE compared with the contralateral side injected with saline. Both effects were dose~ependent and there was a linear relationship betwcen the reduction of iris AChE and papillary diameter . Even in the case of fully developed FAS miosis the eyes responded to light stimulation with further reduction in pupil size . Intravenous administration of atropine immediately reversed the FAS-induced miosis . Chronic animals, injected with 10-200 i+g into the vitreous humor, showed a clear dose-dependent inhibition of iris AChE, up to 21 days after toxin administration . The miosis was maintained during this time in a dosedependent manner and was not reverted by atropine . Preliminary results showed a decrease of the muscarinic receptor population in the treated iris 3 weeks after FAS injection (D . Jerusalinsky, personal communication). These results would indicate that FAS produces a potent and long-lasting inhbition of iris AChE and, a slight down regulation of muscarinic receptor population probably related to the maintained cholinergic hyperactivity. Partially supported by IFS Grant F/1300-1 and IPICS, Uppsala University. StNCe
Cloning and nucleotide sequence of crotamine genes. LnoNnxD A. SMITH and J~~s J. SCHMIDT (Dept. of Toxinology, Pathology Division, U.S . Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701, U.S .A .). A cDNA library was constructed in bacteriophage lambda by using mRNA isolated from the glands of Crotches durissus terriftnu. Primer-initiated reverse transcription created the complement of the mRNA sequences, followed by self-primed second strand synthesis to yield double-stranded cDNAs. A bifunctional oligonucleotide linker was then ligated to the cDNAs prior to insertion into a lambda ORF8 vector and subsequent phage packaging. E. coli MBM7014 was infected with the recombinant molecules, resulting in a primary library containing 1 x 10° pfu per Pg mRNA . Plaques were screened for crotamine by using a nick-translated 200 by fragment encoding myotoxin A and its 3' untranslated region. Over 800 positives were obtained from the first high-density screening of 400,000 plaques. Four of the crotamine clones having insert sizes from 270 to 400 by were chosen and their cDNA inserts subcloned into pGEM-3Z and sequenced. According to the nucleotide sequence from one full-copy gene (26B), its mRNA has the following characteristics . It has a single continuous translational reading frame of 108 nucleotides extending from the first nucleotide sequenced, through an AUG, to a UAA termination colon. Other features include a 5' untranslated region of 13 residues; a 3' nontranslated region of 108 nucleotides, followed by a polyadenylation tract; and a putative polyadenylation signal (AAUAAA) 13 nucleotides upstream from the polyadenylation tract. This mRNA predicts a pretoxin of 65 amino acids, 23 more than the mature toxin. The pretoxin contains a signal sequence 22 residues long and a
2nd International Symposium
839
carboxyl terminal lysine that is subsequently removed. Nucleotide sequence analysis on the clone cDNAs presaged the existence of multiple variants of the crotamine toxin. Even with the different forms identified from the DNA sequences, discrepancies in amino acid sequence with the previously published sequence for crotamine still existed . Purified crotamine was commercially obtained and chromatographed on an FPLC Mono S column . The fractionation yielded five distinct peaks, each of which was subjected to amino acid sequencing . Amino acid sequencing confirmed that each peak was crotamine and also corroborated the DNA sequence obtained previously. Results of the nucleotide and amino acid sequencing will be presented.
Regulation andfunctional implications of concanavalin A (Con A) anda-bungarotoxin (a-BGTJ receptor sites in QmK,z G. W. BOLJRNE' and S. GEERrsaN"(Secretory Process Research adrenal paranewons . J.-M . TRn=ARO,' M. Program, Department of Pharmacology, University of Ottawa, Ottawa and 'McGill University, Montreal, Canada). ExrosuRE of cultured chromaffin cells (CC) to Con A (5 x 10 - '-IO - S M) produced a dose-related and a noncompetitive inhibition of the secretory responses to acetylcholine (ACh) (5 x 10 6-10 ' M) . Studies with fluorescein isothiocyanate~onjugated Con A indicated that after 20 min exposure to Con A, the lectin was taken up by the CC. The uptake of Con A, which was not modified by colchicine (10- 'M) treatment took place without apparently intermediate stage of 'patch' of 'cap' formation. The responses to ACh were promptly restored by a-methyl-D-mannoside in cells exposed to Con A for different periods of time (10 min-8 hr). After 8 hr exposure to Con A, some lectin was still present on CC surface as indicated by experiments with fluorescein isothiocyanate-conjugated immunoglobin G anti-Con A. The secrctory responses to depolarizing concentrations of K* remained unchanged in CC exposed to Con A for different periods of time (20min-8 hr). The results indicate that Con A blocks the cholinergic responses of CC and that either the "Con A-cholinergic receptor complex" or the "surface Con A receptor" which modify the cholinergic responses remains on the CC surface during the uptake of part of the lectin. The results show that internalization of Con A does not interfere with the cellular mechanisms of amine extrusion. Con A also blocked the binding of a-BGT to CC in culture. Furthermore, incubation of cultured CC with d-tubocurarine or mecamylamide for 0.5 to 6 days results in an eight-fold increase in the binding of a-BGT to CC . Hexamethonium and dehydro-ß-erythroidine on the other hand, had no effects. This enhanced a-BGT binding was due to an increase in the number of a-BGT sites without any change in the affinity of the receptors for a-BGT. The increase in a-BGT binding sites produced by the cholinergic antagonists was reversed by either nicotine or carbachol, suggesting that the cholinergic receptor site is important for up-regulation of a-BGT sites. Exposure of CC to either high K* or to protein kinase C stimulants such as phorbol esters increased the number of a-HGT receptor sites on CC surfaces . On the other hand, forskolin and dibutyryl cAMP partially prevented the up-regulation induced by K' and d-tubocurarine . These observations indicate that several mechanisms exist for the regulation of a-BGT binding sites in CC and suggest that this type of receptor population may play an important role in this tissue . Supported by MRC Program Grant PG-20 and MRC MT-7254.
Neurotoxins as neurobiological tools to study nerve-muscle interactions. O. D. LIHITEL (Instituto de Biologis Celular, Facultad de Medicina, U.B .A ., Paraguay 2155, Buenos Aires (1121), Argentina) . SoME frog muscles are made up of different types of muscle fibres like the single-innervated fast fibres, the multiple-innervated brtermedlate 1lbrts, both capable of eliciting action potentials, and the multiple-innervated slow 116res, incapable of generating action potentials . They are innervated by axons of different conduction velocity, fast, intermediate and slow, respectively, and they differ in the characteristics of the synaptic acetylcholine channel, while they share the same type of denervated-induced extrajunctional channels. After denervation or reinnervation by fast axons, slow muscle fibres acquire the ability to generate action potentials . The capability to generate action potentials can also be induced by intramuscular injection of a-bungarotoxin . It is postulated that a-bungarotoxin induces the action potential mechanism because it blocks acetylcholine receptors and thus interferes with the action of non-quantal acetylcholine leaking from the nerve terminals. Slow muscle fibres wen cultured in the presence of a cholinergic agonist to test this hypothesis .
The structure of Mojave toxin as revealed by single crystal X-ray diffraction analysis . KEITH H. WARD,' DOUGLAS M. COLLINE,' VIRGINIA PE7T,' KARL HARDMAN,' TRINA NORDEN,' STEVEN AIRD' and IVAN KAISER2 (Code 6030,
2nd International Symposium
and Glu analyses using RP-HPLC with fluorometric detection after OPA procolumn derivatization .' The basal outflow of Asp/Glu in this preparation was 10-30pmole/min . A significant Ca 2`-dependent increase was observed after 3 min K" 20 mM application. When added to the superfusion medium, DTX significantly increased the release of ASP and GLU. After the initial increase, the amino acid levels gradually returned to basal values . The effect of DTX was partially Cap'-dependent and was not abolished by pretreatment with D-ASP. A dose-dependent effect was scen between 10_ 6 to 10 -9 M DTX. At 7 x 10 - b M, the DTX-evoked release was 300% for Glu and 160% for Asp respect to basal values. After 20 min of continuous DTX superfusion synaptosomes failed to further GLU release when depolarized with 20 mM K" in wntrast with a 600% increase in control microcolumns . These results suggest that DTX induces the release of a vesicular pool of Glu and, to a lesser extent, of Asp, leading to a depletion of the transmitter pool, suggesting that excitatory DTX effects could be related to an increased release of excitatory amino acids in the CNS . This work was partially supported by LP.LC.S. (International Program in Chemical Sciences, Uppsala, Sweden) . F LINDROTH, P. and MorrFrt, K. Analyt . Chem . Sl, 1667 (1979).
Some pharmacological aspects of iris acetylcholinesterase inhibition by jasciculins. R. Savatan, J. MAZZELIA, J. STrextvsct~t~rrtz and F. DN~s (Neurochemistry Div., Instituto de Investigaciones Biologicas Clemente Estable. Montevideo, Uruguay and Laboratory of Glaucoma Research, Pharmacia Ophthalmic AB, Uppsala, Sweden). the cholinergic innervation of the eye participates in several physiological functions, such as the regulation of papillary size, intraocular pressure, ocular circulation, etc., it appeared important to study the role of two peptides, called fasciculins (FAS), recently isolated from the venom of the green mamba snake Dendroaspis angusticeps which have been demonstrated to be powerful inhibitors of acetylcholinesterase (AChE) . Injected in the anterior chamber of rabbits' eyes, FAS (5-30 Ng, total dose) provoked a marked miosis and a significant inhibition (more than 50%) of iris AChE compared with the contralateral side injected with saline. Both effects were dose~ependent and there was a linear relationship betwcen the reduction of iris AChE and papillary diameter . Even in the case of fully developed FAS miosis the eyes responded to light stimulation with further reduction in pupil size . Intravenous administration of atropine immediately reversed the FAS-induced miosis . Chronic animals, injected with 10-200 i+g into the vitreous humor, showed a clear dose-dependent inhibition of iris AChE, up to 21 days after toxin administration . The miosis was maintained during this time in a dosedependent manner and was not reverted by atropine . Preliminary results showed a decrease of the muscarinic receptor population in the treated iris 3 weeks after FAS injection (D . Jerusalinsky, personal communication). These results would indicate that FAS produces a potent and long-lasting inhbition of iris AChE and, a slight down regulation of muscarinic receptor population probably related to the maintained cholinergic hyperactivity. Partially supported by IFS Grant F/1300-1 and IPICS, Uppsala University. StNCe
Cloning and nucleotide sequence of crotamine genes. LnoNnxD A. SMITH and J~~s J. SCHMIDT (Dept. of Toxinology, Pathology Division, U.S . Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701, U.S .A .). A cDNA library was constructed in bacteriophage lambda by using mRNA isolated from the glands of Crotches durissus terriftnu. Primer-initiated reverse transcription created the complement of the mRNA sequences, followed by self-primed second strand synthesis to yield double-stranded cDNAs. A bifunctional oligonucleotide linker was then ligated to the cDNAs prior to insertion into a lambda ORF8 vector and subsequent phage packaging. E. coli MBM7014 was infected with the recombinant molecules, resulting in a primary library containing 1 x 10° pfu per Pg mRNA . Plaques were screened for crotamine by using a nick-translated 200 by fragment encoding myotoxin A and its 3' untranslated region. Over 800 positives were obtained from the first high-density screening of 400,000 plaques. Four of the crotamine clones having insert sizes from 270 to 400 by were chosen and their cDNA inserts subcloned into pGEM-3Z and sequenced. According to the nucleotide sequence from one full-copy gene (26B), its mRNA has the following characteristics . It has a single continuous translational reading frame of 108 nucleotides extending from the first nucleotide sequenced, through an AUG, to a UAA termination colon. Other features include a 5' untranslated region of 13 residues; a 3' nontranslated region of 108 nucleotides, followed by a polyadenylation tract; and a putative polyadenylation signal (AAUAAA) 13 nucleotides upstream from the polyadenylation tract. This mRNA predicts a pretoxin of 65 amino acids, 23 more than the mature toxin. The pretoxin contains a signal sequence 22 residues long and a
2nd International Symposium
839
carboxyl terminal lysine that is subsequently removed. Nucleotide sequence analysis on the clone cDNAs presaged the existence of multiple variants of the crotamine toxin. Even with the different forms identified from the DNA sequences, discrepancies in amino acid sequence with the previously published sequence for crotamine still existed . Purified crotamine was commercially obtained and chromatographed on an FPLC Mono S column . The fractionation yielded five distinct peaks, each of which was subjected to amino acid sequencing . Amino acid sequencing confirmed that each peak was crotamine and also corroborated the DNA sequence obtained previously. Results of the nucleotide and amino acid sequencing will be presented.
Regulation andfunctional implications of concanavalin A (Con A) anda-bungarotoxin (a-BGTJ receptor sites in QmK,z G. W. BOLJRNE' and S. GEERrsaN"(Secretory Process Research adrenal paranewons . J.-M . TRn=ARO,' M. Program, Department of Pharmacology, University of Ottawa, Ottawa and 'McGill University, Montreal, Canada). ExrosuRE of cultured chromaffin cells (CC) to Con A (5 x 10 - '-IO - S M) produced a dose-related and a noncompetitive inhibition of the secretory responses to acetylcholine (ACh) (5 x 10 6-10 ' M) . Studies with fluorescein isothiocyanate~onjugated Con A indicated that after 20 min exposure to Con A, the lectin was taken up by the CC. The uptake of Con A, which was not modified by colchicine (10- 'M) treatment took place without apparently intermediate stage of 'patch' of 'cap' formation. The responses to ACh were promptly restored by a-methyl-D-mannoside in cells exposed to Con A for different periods of time (10 min-8 hr). After 8 hr exposure to Con A, some lectin was still present on CC surface as indicated by experiments with fluorescein isothiocyanate-conjugated immunoglobin G anti-Con A. The secrctory responses to depolarizing concentrations of K* remained unchanged in CC exposed to Con A for different periods of time (20min-8 hr). The results indicate that Con A blocks the cholinergic responses of CC and that either the "Con A-cholinergic receptor complex" or the "surface Con A receptor" which modify the cholinergic responses remains on the CC surface during the uptake of part of the lectin. The results show that internalization of Con A does not interfere with the cellular mechanisms of amine extrusion. Con A also blocked the binding of a-BGT to CC in culture. Furthermore, incubation of cultured CC with d-tubocurarine or mecamylamide for 0.5 to 6 days results in an eight-fold increase in the binding of a-BGT to CC . Hexamethonium and dehydro-ß-erythroidine on the other hand, had no effects. This enhanced a-BGT binding was due to an increase in the number of a-BGT sites without any change in the affinity of the receptors for a-BGT. The increase in a-BGT binding sites produced by the cholinergic antagonists was reversed by either nicotine or carbachol, suggesting that the cholinergic receptor site is important for up-regulation of a-BGT sites. Exposure of CC to either high K* or to protein kinase C stimulants such as phorbol esters increased the number of a-HGT receptor sites on CC surfaces . On the other hand, forskolin and dibutyryl cAMP partially prevented the up-regulation induced by K' and d-tubocurarine . These observations indicate that several mechanisms exist for the regulation of a-BGT binding sites in CC and suggest that this type of receptor population may play an important role in this tissue . Supported by MRC Program Grant PG-20 and MRC MT-7254.
Neurotoxins as neurobiological tools to study nerve-muscle interactions. O. D. LIHITEL (Instituto de Biologis Celular, Facultad de Medicina, U.B .A ., Paraguay 2155, Buenos Aires (1121), Argentina) . SoME frog muscles are made up of different types of muscle fibres like the single-innervated fast fibres, the multiple-innervated brtermedlate 1lbrts, both capable of eliciting action potentials, and the multiple-innervated slow 116res, incapable of generating action potentials . They are innervated by axons of different conduction velocity, fast, intermediate and slow, respectively, and they differ in the characteristics of the synaptic acetylcholine channel, while they share the same type of denervated-induced extrajunctional channels. After denervation or reinnervation by fast axons, slow muscle fibres acquire the ability to generate action potentials . The capability to generate action potentials can also be induced by intramuscular injection of a-bungarotoxin . It is postulated that a-bungarotoxin induces the action potential mechanism because it blocks acetylcholine receptors and thus interferes with the action of non-quantal acetylcholine leaking from the nerve terminals. Slow muscle fibres wen cultured in the presence of a cholinergic agonist to test this hypothesis .
The structure of Mojave toxin as revealed by single crystal X-ray diffraction analysis . KEITH H. WARD,' DOUGLAS M. COLLINE,' VIRGINIA PE7T,' KARL HARDMAN,' TRINA NORDEN,' STEVEN AIRD' and IVAN KAISER2 (Code 6030,