Chemical modification of histidine and lysine residues of crotoxin

Jeng, Tzyy‐Wen; Fraenkel‐Conrat, H.

doi:10.1016/0014-5793(78)80354-8

Cited by 40 publications

(12 citation statements)

References 21 publications

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“…In agreement with the results of Jeng and Fraenkel-Conrat [27] we observed that p-bromophenacyl bromide irreversibly inhibits the phospholipase activity of component B by alkylating one of its two histidine residues (data not shown). We found that this reaction was prevented by substrates under conditions which allow the catalytic activity of component B: detergent-solubilized phosphatidylcholine afforded a full protection while in the micellar state it produced only a marginal effect.…”

Section: Irreversible Inhibitionsupporting

confidence: 81%

“…We found that this reaction was prevented by substrates under conditions which allow the catalytic activity of component B: detergent-solubilized phosphatidylcholine afforded a full protection while in the micellar state it produced only a marginal effect. As also shown by Jeng and Fraenkel-Conrat [27], the crotoxin complex was totally insensitive to the alkylating agent, suggesting that the histidine residue of the active site of component B is occluded by the non-catalytic component A. We found, however, that an inactivated component Acomponent B complex could be prepared by reassociating the alkylated component B with native component A.…”

Section: Irreversible Inhibitionmentioning

confidence: 50%

“…In the first series of experiments we used an irreversible inhibitor, p-bromophenacyl bromide, which specifically alkylates a histidine residue and thereby suppresses the catalytic activity of component B [27]. Crotoxin itself does not react with the inhibitor, but we found that alkylated component B recombines with component A, although with a reduced affinity.…”

Section: Resultsmentioning

confidence: 99%

“…In this case, as observed with b-bungarotoxin [25], the time required for crotoxin to block the neuromuscular transmission is prolonged [26]. In addition, Jeng and Fraenkel-Conrat [27] have shown that the toxicity of crotoxin component B is abolished after irreversible inhibition of its catalytic activity by the alkylating inhibitor, p-bromophenacyl bromide [21, 28-301. Therefore, as observed for other neurotoxins [20,21,29,31,32] it appears that the pharmacological action of crotoxin is exerted at least in part, through its phospholipase activity.…”

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confidence: 92%

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Relationship between the Pharmacological Action of Crotoxin and Its Phospholipase Activity

Marlas¹,

Bon²

1982

European Journal of Biochemistry

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Crotoxin, the major neurotoxin from Crotalus durissus terrificus venom, blocks the postsynaptic response of Electrophorus electroplaque to bath-applied carbamoylcholine. Crotoxin is composed of two non-covalently linked subunits: an acidic non-enzymatic component, A, and a basic phospholipase component, B. According to our previous studies, component A restricts the binding of component B to high-affinity sites which occur in Torpedo membrane fragments in approximatively equivalent amount to the cholinergic receptor sites. We demonstrate here that the catalytic activity of component B is actually required for the postsynaptic blocking action of the toxin.Irreversible inactivation of component B was achieved by alkylation with p-bromophenylacyl bromide of a histidine residue in the catalytic site. Although the native crotoxin complex was insensitive to the inhibitor, suggesting that the catalytic center is protected by component A, we obtained an enzymatically inactive crotoxin by recombination of the alkylated component B with component A. This modified crotoxin was devoid of enzymic activity, toxicity and pharmacological activity on the electroplaque.The phospholipase activity of component B may be reversibly inhibited by replacing Ca2+ with Sr2+ ions in the physiological solution. This allowed us to identify several steps in the action of the toxin: a quasi-irreversible binding step which occurs either in the presence of Ca2+ or Sr2+ ions, and a catalytic step which requires Ca2+ ions and is responsible for the irreversible blockade of the electroplaque response to cholinergic agonists.Finally, we examined the effect of various toxic and non-toxic phospholipases on the electroplaque response. The data exclude that the postsynaptic blockade results from indiscriminate hydrolysis of phospholipidb. They rather suggest that the pharmacological efficiency of crotoxin is due to the fact that component B exerts its catalytic activity at critical postsynaptic sites. These conclusions are in excellent agreement with the explanation of the synergistic effect of the two subunits which we derived from our binding data. The postsynaptic sensitive sites for crotoxin action might be closely related to the acetylcholine receptor sites.Crotoxin, the major toxic component in the venom of the brazilian rattlesnake, Crotalus durissus terrificus, was the first snake neurotoxin to be isolated [I] (for review see [2]). It is a complex of two different subunits [3 -51 : a basic protein which carries the phospholipase activity, component B (Mr 12000), and an acidic component, component A or crotapotin ( M , 10000) which has no known catalytic properties and no toxicity by itself, but enhances the toxicity of component B Crotoxin has been shown to cause a peripheral paralysis in vertebrates by blocking the neuromuscular transmission [9-121. An early observation that the response of denervated rat diaphragm to acetylcholine was decreased after exposure to the toxin suggested a postsynaptic action [I 0,11,13]. However a detailed electro...

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Section: Irreversible Inhibitionsupporting

confidence: 81%

Section: Irreversible Inhibitionmentioning

confidence: 50%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 92%

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Relationship between the Pharmacological Action of Crotoxin and Its Phospholipase Activity

Marlas¹,

Bon²

1982

European Journal of Biochemistry

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“…Consequently, the synaptic vesicle cycle may be blocked irreversibly at the ultimate stage of endocytosis, as the vesicle neck closure is interrupted [10]. Although a direct correlation between enzymic activity and lethal potency of neurotoxic PLA # s has not been observed [11], it was shown a long time ago that enzymic activity is essential for full expression of the neurotoxic effect [12]. In spite of numerous attempts to identify the surface residues of different PLA # neurotoxins involved in the complex process of presynaptic neurotoxicity, the molecular basis of their toxicity has yet to be resolved [5].…”

Section: Introductionmentioning

confidence: 99%

Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity

Petan

Križaj

Gubenšek

et al. 2002

Biochemical Journal

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Ammodytoxins (Atxs) are group II phospholipases A2 (PLA2s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA2 toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe24, in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp24 mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40–80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA2 activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe24 also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn24 mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe24 in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.

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