Judith K. Marquis

Noncompetitive inhibition by aluminum, scandium and yttrium of acetylcholinesterase from Electrophorus electricus

Measurements of altered activity of soluble acetylcholinesterase from E. electricus electric organ by the inorganic cations aluminum, scandium and yttrium demonstrate that these ions are noncompetitive enzyme inhibitors. Al3+ inhibited enzyme activity at all substrate and inhibitor concentrations studied. Inhibition by Al3+ did not appear to be sensitive to the active site-specific, competitive ligand physostigmine or to calcium, a peripheral site-binding activator cation. Inhibition by another peripheral site-binding noncompetitive inhibitor, decamethonium, was not altered by Al3+. Al3+ appears thus to have interacted with a class of peripheral anionic sites on AChE distinct from the beta- or P1 peripheral anionic sites that bind Ca2+ and C-10 and may be a useful probe of a subclass of gamma- or P2-4 peripheral anionic sites. A possible mechanism for Al3+ neurotoxicity, via alterations of the enzymes of cholinergic neurotransmission, is also suggested.

The effect of electrical stimulation on the action of sulfhydryl reagents in the giant axon of squid; Suggested mechanisms for the role of thiol and disulfide groups in electrically-induced conformational changes

SummaryConduction block by thiol reagents is potentiated by repeated, brief electrical stimulation. These studies have been quantitated with N-ethylmaleimide and mercurochrome showing a nonlinear relationship between dose and number of stimuli required to produce inexcitability.p-Chloromercuribenzoate, mercurochrome and fluorescein mercuric acetate block conduction and are reversible with β-mercaptoethanol and exhibit the “stimulation effect.” N-Ethylmaleimide, Ellmans reagent (DTNB), and 2-dimethylaminoethyl selenolbenzoate exhibit the “stimulation effect”, but blockade is irreversible. In a series of local anesthetics, procaine, 2-diethylaminoethyl thiolbenzoate, and 2-dimethylaminoethyl selenolbenzoate, only the selenolester reacts with SH groups and shows a “stimulation effect”. Iodoacetate and iodoacetamide block nerve conduction without a stimulation effect. Possible interpretations of this effect include: altered permeability, unmasking of buried SH groups in the membrane, or electrolytic reduction of disulfides.

Direct binding studies of 125I-α-bungarotoxin and 3H-quinuclidinyl benzilate interaction with axon plasma membrane fragments. Evidence for nicotinic and muscarinic binding sites

Abstract The attachment of 125 I-α-bungarotoxin (BgTx) which is reportedly bound exclusively to “nicotinic” acetylcholine receptors, as well as 3 H-atropine and 3 H-3-quinuclidinyl benzilate (QNB), which reportedly bind exclusively to “muscarinic” receptors, was measured in isolated lobster axon plasma membrane fragments and in the soluble axonal protein fraction. 125 I-α-BgTx binding was also measured in lysolecithin-solubilized fragments. Binding assays were adapted for these studies and are described in detail. High affinity, saturable binding of all three ligands to membrane fragments was observed, as well as binding of BgTx to a macromolecule present in both the soluble fraction and the membrane fragments. These experiments provide the first evidence for the very tight binding of both “nicotinic” and “muscarinic” ligands in peripheral nerve.

Local anesthetics noncompetitively inhibit terbium binding to the exterior surface of nerve membrane vesicles

It has previously been shown that terbium binds to membrane vesicles prepared from the walking leg nerve of the lobster (Homarus americanus) with a high affinity Kd of 2.2 microM. Fluorescence of bound Tb3+ occurs via energy transfer from the aromatic residues of proteins (gamma ex = 280 nm; gamma em = 546 nm), and calcium inhibits Tb3+ binding competitively with a Ki of 1.8 mM. Displacement studies with EDTA demonstrate that more than 95% of the bound Tb3+ is at the vesicle exterior and is not being taken up by the vesicles. To investigate the putative role of Ca2+ in the interaction of local anesthetics with axonal membranes, lidocaine and the analogs GX-HCl and QX-314 were tested as inhibitors of Tb3+ binding. Inhibition by lidocaine is seen only at considerably higher doses (25 mM) than are required for conduction block of intact nerves (5 mM). Inhibition by lidocaine and the primary amine analog GX-HCl is entirely noncompetitive, whereas the quaternary ammonium derivative QX-314 appears to be a mixed competitive-noncompetitive inhibitor of Tb3+ binding. These data are not compatible with the hypothesis that there is a functionally essential cation binding site on the axonal membrane surface for which Ca2+ and local anesthetics compete, although local anesthetic action may be modified indirectly by altered calcium concentrations. Evidence is presented for a mechanism by which local anesthetics indirectly displace Tb3+ by altering the physical state of the axonal membrane.

The binding of thiol reagents to axonal membranes: The effect of electrical stimulation

Summary Fluorescence techniques have been used to measure the binding of mercurochrome to walking leg nerve bundles of the spider crab. Reversibility of binding of mercurochrome by 2-mercaptoethanol was also measured. Both parameters were increased by electrical stimulation of the nerve bundle.

On the Specificity of 125I-α-Bungarotoxin Binding to Axonal Membranes

Abstract: 125I‐α‐Bungarotoxin (α‐BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of α‐BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible (KDapp± 1.7 ± 0.32 × 10‐7 M; Bmax± 249 ± 46 pmol/mg protein) at pH 7.8, 10 mM‐Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca2+ and Mg2+) in the medium. 125I‐α‐BGT binding could be inhibited by cholinergic drugs (atropine ≅d‐tubocurarine > nicotine > carbamylcholine ≅ choline) and local anesthetics (procaine > tetracaine = lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4‐aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to α‐BGT binding sites in other tissues are discussed.

Interactions of lanthanum with purified and intact cell acetylcholinesterase of Electrophorus electricus.

SummaryThe effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl3 produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl3, initial activity was markedly increased. LaCl3 exerted a marked, concentration-dependent inhibition of intact cell AChE.La3+ and Ca2+ appear to interact competitively. In the presence of both 10mm CaCl2 and 0.1mm LaCl3, the initial activity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 × 10−4m. Inhibition of intact cell enzyme by 0.1mm LaCl3 was relieved by increasing the CaCl2 concentration to 10mm at ACh concentrations less than 2 × 10−4m.The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.

Concanavalin A-Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites

Abstract: Lysolecithin‐solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)‐sepharose. Bound glycoproteins were eluted with α‐methyl‐D‐ mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A‐binding glycoprotein fraction. A 5‐fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [3H]quinuclidinyl benzilate (QNB), [3nicotine (Nic), and [1251]α‐bung arotoxin (BgTx) in an attempt to distinguish possible “muscarinic” and “nicotinic” binding sites in axonal membranes. All of the high‐affinity “muscarinic” [3H]QNB binding activity appeared in the non‐Con A‐binding protein fractions, while binding of the two “nicotinic” ligands, [3Nic and 125I‐BgTx, was found in both the glycoprotein and non‐Con A‐binding protein fractions. BgTx interaction with the Con A‐binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non‐Con A‐binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.

The effects of calcium and lanthanum on the interaction of decamethonium with soluble acetylcholinesterase from Electrophorus electricus

THERE is considerable evidence supporting a regulatory role for non-catalytic anionic sites on the AChE (EC 3.1.1.7) molecule. Early evidence for the presence of negative sites distinct from the active centre was presented by BERCMANN et al. (1950) and later by WILSON (1960). CHANCEUX et al. (1968) measured 25G300 ACh molecules bound per molecule of AChE in the presencc of active site inhibitors such as eserine, supporting the notion of a large number of non-catalytic substrate-binding sites. Topographically-distinct non-catalytic sites were also postulated by KUHNEN (1972) in studies on the inhibition of AChE by his-pyridinium compounds. Further characterization of a peripheral anionic site has been obtained with 10-carbon bis-quaternary ammonium ligands where the quaternary nitrogens are separated by 14w. CHANGEUX (1966) demonstrated that decamethonium (C, O)r a his-quaternary ammonium ligand which produces a depolarizing block of cholinergic receptors, inhibits a solubilized Torpedo AChE preparation. ROUFOGALIS & QUIST (1972) obtained similar results with erythrocyte AChE and suggested that C , , is bound in a bridge-like manner to both the active site and to peripheral anionic sites. More recently. TAYLOR & LAPPI (1975) have shown that C,,, displaces the fluorescent probe, propidium diiodide, from a peripheral anionic centre on purified Torpedo AChE. These findings support previous work indicating the existence of two discrete anionic sites on AChE (BELLEAU et al. (1970); KITZ et al. (1970; WOMBACH~R & WOLF

A reevaluation of calcium-local anesthetic antagonism

Abstract Although it has been shown that Ca 2+ can inhibit the conduction blocking action of local anesthetic agents in lobster peripheral nerve, it is not known whether Ca 2+ directly competes for anionic membrane sites or indirectly alters the drug-membrane interaction. The present experiments were carried out during a sufficiently long time course to determine whether the blocking action of lidocaine, procaine, or tetracaine was reduced by Ca 2+ or whether Ca 2+ only delayed the onset of local anesthesia. Lidocaine HCl (5 m m ) reduced the amplitude of the compound action potential by 50% in 10 min in low-Ca 2+ (5 m m ) lobster physiologic saline, whereas in high-Ca 2+ (75 m m ) saline, 5 m m lidocaine reduced the action potential amplitude 50% only after 25 min external bath perfusion. Similar results were seen with 2 m m procaine and 0.2 m m tetracaine. The data confirm the suggestion that Ca 2+ merely slows the passage of local anesthetic agents across glial membranes and are not compatible with a model that describes competition of Ca 2+ and local anesthetic agents for a specific binding site in nerve membranes.