Fasciculins, anticholinesterase toxins from the venom of the green mamba Dendroaspis angusticeps

Two toxins that are potent inhibitors of acetylcholinesterase have been isolated from the venom of the green mamba, Dendroaspis angusticeps. The toxins have been called fasciculins since after injection into mice (i.p. 0.5-3 micrograms/g body weight) they cause severe, generalized and long-lasting (5-7 h) fasciculations. Homogenates of diaphragm, tibialis anterior and gastrocnemius muscles from mice injected with fasciculins showed a decrease in acetylcholinesterase activity by 45-60% compared to muscles from control animals. Histochemical staining revealed a greatly reduced acetylcholinesterase activity at neuromuscular junctions. Fasciculins have 61 amino acid residues and four disulfides. The molecular weights are 6765 (fasciculin 1) and 6735 (fasciculin 2). The sequences of the two toxins differ probably only at one position by a replacement of Tyr with Asp/Asn. 1 g of venom contained about 40 mg of fasciculins, 2/3 of which was fasciculin 2. A similar inhibitor has also been isolated from D. polylepis (black mamba) venom. The sequence of fasciculin 2 is known. Most of the positive charges are concentrated in a small section of the central part of the molecule, and most of the negative charges are in the C-terminal region. Fasciculins appear to have a pronounced dipole character. Fasciculin binds to the peripheral anionic site, since it can displace propidium, a probe for that site, from acetylcholinesterase. In vitro, in Krebs-Henseleit solution containing 2 mM NaH2PO4 (pH 7.4), fasciculin 2 inhibits acetylcholinesterase from human erythrocytes (Ki = 1.1 X 10(-10) M, 37 degrees C), rat muscle (Ki = 1.2 X 10(-10) M, 37 degrees C) and Electrophorus electricus (Ki = 3.0 X 10(-10) M, 22 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Aging and rat brain muscarinic receptors as measured by quinuclidinyl benzilate binding

Measurement of cholinergic muscarinic receptor binding in various rat brain areas using the ligand [³H]quinuclidinyl benzilate indicates that receptor binding is decreased in striatum and cerebellum of aged female rats (22 months old) as compared to younger rats (4 months old). Decreases were not observed in cortex, hippocampus, hypothalamus, or amygdala areas. Further examination of [³H]quinuclidinyl benzilate binding in subcellular fractions of aged and young rat cerebellum and striatum indicated a decrease in binding in the crude nuclear and crude synaptosomal fractions. Binding data indicate the observed decrease in specific ligand binding is due to a decrease in number of binding sites while receptor affinity does not appear to change.Supported by the Research Service of the Veterans Administration and by Research Grant NS 13227 from NINCDS.     

A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 40 mM KCl with a potency (K0.5 = 20 nM) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 100 nM) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (K0.5 = 3 nM) and DNP (K0.5 = 3 nM) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cGMP responses to CNP (K0.5 = 10 nM) than to ANP or DNP (K0.5 = 100 nM). Finally, DNP completely prevented the specific 125I-ANP binding to clearance receptors in cultured aortic myocytes with a potency (Kd = 10 nM) that was less than that of ANP (Kd = 0.3 nM). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors.     

Effect of lysophasphatidylcholine on sensitivity of the heart to acetylcholine and binding of quinuclidinyl benzilate to myocardial membranes

Chemical nature of the blood serum factor exerting cholinolytic effect on the animal heart, was determined. The effect is due to the serum lipid component: lysophosphatidylcholine. The latter in concentration 1-25 microM suppresses the sensitivity of the frog perfused heart ventricle to acetylcholine. Phosphatidylcholine was found to protect the heart from the lysophosphatidylcholine effect. Lysophosphatidylcholine also affected the binding of the acetylcholine high affinity antagonist [3H]-quinuclidinylbenzilate to the rabbit atria cell membranes. The mechanism of this effect is related to an increased ability of muscarinic receptors to form oligomeric complexes in presence of lysophosphatidylcholine, the latter manifesting a positive cooperativity on binding to ligand.     

Structure-binding relationship of quinuclidinyl benzilate analogs on N4TG1 neuroblastoma muscarinic receptors

By Scatchard plot analysis of [³H]QNB (quinuclidinyl benzilate) binding, there are 2×10⁵ muscarinic sites/cell with aK _d about 10 nM in N4TG1 neuroblastoma cells. We have now examined a group of compounds structurally related to aprophen and QNB for their ability to compete with the binding of QNB to the muscarini receptor. Using this structure-inhibition relationship, the functional groups of the muscarinic ligand necessary for binding were partially characterized. It was found that the quinuclidinyl ring structure of QNB can be substituted by either alkane, H, or pyrrolidine at the N without loosing their ability to bind. The addition to the quinuclidinyl ring increases the bulk of the structure and decreases binding. Like the benzilate in QNB, a similar hydrophobic structure is apparently required for the binding.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Purification and partial characterization of K+ channel blockers from the venom of Dendroaspis angusticeps.

Two polypeptides (designated DTX-A and DTX-B) were purified from crude snake venom of Dendroaspis angusticeps using gel filtration, cation exchange colum chromatography and cation exchange high performance liquid chromatography, and their blocking actions of K⁺ channels were investigated in rat brain synaptosomes. Both DTX-A and DTX-B inhibited the voltage-dependent ⁴²K efflux from the synaptosomes. DTX-A blocked ⁴²K efflux of both the rapidly inactivating phase (component T) and the slowly inactivating phase (component S). The inhibitory effect of DTX-A on component T was pronounced compared with that on component S. However, DTX-B selectively blocked ⁴²K efflux of component S. The molecular weights of DTX-A and DTX-B were estimated to be ca 10,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of these toxins is different from that of polypeptide purified from the venom of D. angusticeps (-, β, γ- and δ-DTX). These results suggest that DTX-A and DTX-B are new polypeptides which block voltage-dependent K⁺ channels selectively, and that they are useful tools for investigating the K⁺ channel.     

N-substituted derivatives of 4-piperidinyl benzilate: affinities for brain muscarinic acetylcholine receptors

N-Substituted derivatives of 4-piperidinyl benzilate were synthesized and their affinities for central muscarinic cholinergic receptors determined using an in vitro radioligand binding assay. 4-Piperidinyl benzilate exhibited a Ki value of 2.0 nM. N-Substitution with a methyl or an ethyl group increased the affinity to 0.2 nM, whereas substitution with a n-propyl or isopropyl group decreased the binding affinity over 100 fold. Compounds with aralkyl substitutions at the nitrogen atom of piperidinyl benzilate were also synthesized and evaluated. The Ki values (nM) obtained for these compounds were: benzyl, 0.2; p-nitrobenzyl, 13.0; p-fluorobenzyl, 3.0; phenethyl, 8.0; p-nitrophenethyl, 15.0. These data suggest that a binding region near the piperidinyl nitrogen may tolerate bulky aromatic substitutions (e.g., benzyl or phenethyl) as well or better than straight chain or branched alkyl substitutions (e.g., n-propyl or isopropyl).     

Effect of aging on the interaction of quinuclidinyl benzilate,N-methylscopolamine,pirenzepine, and gallamine with brain muscarinic receptors

The objective of the present study was to investigate the effects of senescence on the binding characteristics of muscarinic receptors by using [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]N-methylscopolamine ([³H]NMS) as ligands in young (3months), middle-age (10months) and old (24 months) male Fischer 344 rats. Muscarinic receptor density was found to decrease significantly with aging in certain brain regions, depending on the ligand employed. Moreover, the relative proportions of M₁ and M₂ muscarinic receptor subtypes was not significantly altered by aging, except in the aged striatum. Furthermore, the dissociation kinetics of [³H]NMS in the cerebral cortex and their allosteric modulation by gallamine were only slightly influenced by age.     

Solubilization by various detergents, of the muscarinic cholinoreceptor and its complex with quinuclidinyl benzilate

The possibilities to solubilize the rat brain cortex muscarinic acetylcholine receptor and its complex with [3H]-L-quinuclidinyl benzilate (QNB) were studied, using 14 detergents. It was shown that the native muscarinic cholinoreceptor was solubilized in addition to digitonin, also by CHAPS, with a 6% yield. Besides, the receptor-QNB complex was solubilized with the detergents Triton X-100, -102, -114, -165 (with 30% and 50% yields) and within a narrow concentration range with sodium dodecyl sulfate (50% yield). Some detergents of the Tween series, e.g., Triton X-45 and -305, as well as sodium deoxycholate and sodium oxycholate, did not solubilize the native receptor and its complex with QNB. It was found that yield of receptor solubilization did not exceed half of the total number of the receptor sites in the membranes, despite the fact that different concentrations of detergents were applied. The solubilization yield did not increase, when different mixtures of detergents were used. It was assumed that incomplete solubilization of the receptor protein reflects its heterogeneity in the membrane structure.     

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M₁ receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [³H]N-methylscopolamine binding to cloned muscarinic M₁ and M₄ receptors, and reduced binding to M₅ subtype with lower affinity, while they reversibly inhibited the binding of [³H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M₁ receptors at a site distinct from the classical one, and to have affinity for some -adrenoceptors.     

Failure of gallamine and pancuronium to inhibit selectively (-)-[3H]quinuclidinyl benzilate binding in guinea-pig atria

Binding of (-)-[3H]quinuclidinyl benzilate (QNB) to muscarinic sites in guinea-pig atrial and ileal longitudinal muscle homogenates showed the presence of a single population of binding sites in atria (KD = 41 (32-53) (95% confidence limits) pM; Bmax = 0.225 +/- 0.02 pmol/mg protein (3)) and two binding sites in the ileum (KD = 20.9 (8.8-49) pM and 11.3 nM; Bmax = 0.436 +/- 0.09 and 11.85 +/- 2.63 pmol/mg protein (4), respectively). Atropine, gallamine, and pancuronium displaced (-)-[3H]QNB binding from the high affinity binding sites in the two tissues in a dose-dependent manner with -log Ki values of 8.6, 6.4, and 6.9, respectively, in atria and 8.7, 6.8, and 6.9, respectively, in ileal longitudinal muscle. The lack of selectivity of gallamine and pancuronium in binding experiments differed from results obtained in isolated tissue experiments where these antagonists showed a marked difference in their ability to antagonize cholinomimetics in the two tissues. In addition, the Ki values for gallamine and pancuronium in ileal homogenates were ca. 130- and 16-fold lower, respectively, than their KB values determined from isolated tissue experiments. Attempts to correlate data from binding experiments and isolated tissue experiments using combinations of antagonists led to variable results attributed to differences in the rates of dissociation of the antagonists from muscarinic receptors. It is concluded that the interaction of gallamine or pancuronium with agonists or antagonists at muscarinic receptors is not a simple bimolecular interaction.     

Agonists differentiate muscarinic receptors that inhibit cyclic AMP formation from those that stimulate phosphoinositide metabolism

Muscarinic receptor stimulation elicits two distinct biochemical responses in embryonic chick heart cells: inhibition of catecholamine-stimulated cyclic AMP formation and stimulation of phosphoinositide (PhI) hydrolysis. We observe two major differences in the effects of agonists on these responses. First, carbachol and oxotremorine both inhibit cyclic AMP formation, but only carbachol stimulates PhI hydrolysis. Second, the dose-response relationships for the cyclic AMP and PhI responses differ; the half-maximal concentrations of carbachol needed to inhibit cAMP accumulation and stimulate PhI hydrolysis are 2 X 10(-7) and 2 X 10(-5) M, respectively. We carried out radioligand binding studies on intact chick heart cells to determine whether these data could be explained in terms of different agonist binding states of the muscarinic receptor. In intact cells, carbachol competes for [3H]quinuclidinyl benzilate-binding sites with high and low affinity, while oxotremorine shows only high affinity binding. We suggest that the receptor state common to both agonists is the state associated with inhibition of adenylate cyclase, while the very low affinity binding site seen only with carbachol is associated with the PhI response. We also consider the possibility that both responses are caused by a single receptor state that is efficiently coupled to adenylate cyclase inhibition and inefficiently coupled to PhI hydrolysis. Whichever mechanism is correct, our findings demonstrate that muscarinic receptors coupled to adenylate cyclase and the PhI response can be differentiated by virtue of their sensitivity to agonist and the efficiency with which some agonists induce receptor change and elicit receptor-mediated biochemical responses.     

Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.     

Crystals of fasciculin 2 from green mamba snake venom. Preparation and preliminary x-ray analysis

Fasciculin 2 from the venom of the green mamba, Dendroaspis angusticeps, has been crystallized. The crystals are tetragonal, with unit cell dimensions a = 48.9 A and c = 82.0 A, space group P 41 21 2 or P 43212. Density measurements and pseudocentering of the hko zone indicate that there are 16 molecules in the unit cell.     

Multiple binding states of muscarinic acetylcholine receptors in membranes from neuroblastoma X glioma hybrid cells

Membranes of neuron-like NG108-15 hybrid cells bind [³H]quinuclidinyl benzilate (QNB) with high affinity and specificity. Greater than 90% of total [³H]QNB binding is to sites having the pharmacological specificity of muscarinic acetylcholine receptors. Three significant features characterize the interaction of ligands with these sites: (1) Specific binding of [³H]QNB at equilibrium follows a simple adsorption isotherm with an apparent K_D of 1 × 10^?10 M; (2) Rates of [³H]QNB association and dissociation are biphasic and, as the binding reaction proceeds, the fraction of readily dissociable [³H]QNB decreases; (3) Competition against [³H]QNB for specific binding sites by antagonists gives a slope of 1 when analyzed on Hill plots, but competition for binding sites by agonists gives a slope of less than 1. A simple two-step model for activation is proposed to account for these features.