Henry G. Mautner

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.

Evidence for a thiol reagent inhibiting choline acetyltransferase by reacting with the thiol group of coenzyme a forming a potent inhibitor

Abstract Evidence is presented for the thiol reagent methyl methanethiolsulfonate inhibiting choline acetyltransferase (EC 2.3.1.6), not by reaction with an enzymic thiol group, but by reaction with the thiol group of CoA. The resulting CoA methyl disulfide is a potent inhibitor of this enzyme. Its action is reversed competitively by acetyl CoA.

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.

Radioactivation as a method for preparing 75Se-labelled selenium compounds

Chemically pure 6-selenoquinone, 6-selenopurine, and selenocystine were activated by irradiation in the watercooled compartment of a graphite reactor at a neutron flux of 7.5 x 10/sup 11/ neutrons/cm/sup 2/sec for 62 hr. After irradiation the Se/sup 75/ content of the compounds was determined by use of a gamma scintillation chamber. No evidence was found of decomposition during the activation procedure. (C.H.)

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.

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.

Intestinal transport of cystine analogues.

FOR the intestinal transport of amino-acids, the carboxyl and amino-groups appear to be of key importance1. In the case of cystine, there has been suggestive evidence that the disulphide group might also play a part in the movement of the compound2,3. We present here simple but compelling data on the participation of the disulphide moiety in cystine transport. There is shown to be in vitro intestinal transport of cystine and homocystine against a concentration gradient, while 9 analogues which do not possess the —S—S— linkage are not transported.

A direct approach to the determination of the rotational barriers of the acetylcholine and choline molecules

Abstract The temperature dependence of the N.M.R. spectra of choline and acetylcholine as well as those of their β-dideutero analogs was investigated. While broadening of the β-methylene signals was observed at −90°, the coalescence temperature was still not reached at −110°. From this information an upper limit of the rotational barriers around the central methylene carbons of 7.9 kcal could be calculated.