Alkylation of Free Sulfhydryls Fortifies Electroplax Subsynaptic Structures

Abstract

The cysteine-rich 43,000-dalton peripheral membrane protein, nu 1, is localized at the cytoplasmic face of electroplax and muscle cholinergic synapses, where it is thought to play an important role in the endplate supramolecular structure. The peripheral membrane protein properties of nu 1 are inferred by its removal from nicotinic cholinergic membranes by the action of mild alkali or lithium diiodosalicylate. An interesting property of nu 1 is its high concentration of free sulfhydryl groups, whose exact role in synaptic structure is still largely unknown. Alkylation of free sulfhydryls with N-ethylmaleimide (3 mM) has a profound effect on the association of nu 1 with synaptic membranes, rendering nu 1 unextractable by pH 11 treatment or by lithium diiodosalicylate and, concomitantly, decreasing nu 1's electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels. Iodoacetamide and iodoacetate have similar effects, but at concentrations 10- to 100-fold higher than required for N-ethylmaleimide. Furthermore, sulfhydryl modification also stabilizes the association of nicotinic receptor subunits with the detergent-insoluble cytoskeleton. N-Ethylmaleimide treatment increases the fraction of insoluble receptor molecules on extraction with Triton X-100, sodium cholate, or octylglucoside. These results suggest an important role of sulfhydryl groups in the structural stability of the postsynaptic cholinergic membrane.

In Vitro and in Vivo Redox States of the Escherichia coli Periplasmic Oxidoreductases DsbA and DsbC

“…Missiakas et al (13) used iodoacetamide to trap the cysteines while we use a maleimide reagent. Maleimides tend to be more cysteine-specific and faster reacting with cysteines than the haloacetates (38)(39)(40)(41) although microenvironmental factors can greatly influence thiol reactivity and specificity (20,42). Additionally, the three redox states of DsbC cannot be differentiated with iodoacetamide trapping followed by SDS-PAGE.…”

Synaptic Cysteine Sulfhydryl Groups as Targets of Electrophilic Neurotoxicants

“…Binding of acetylcholine or the plant alkaloid, nicotine, to the nAChR increases the channel conductance of most cations, although monovalent ions (Na þ , K þ ) are preferred. Several studies have shown that agonist affinity, ion conductance, and structural stability of the nAChR are dependent upon the redox state of protein sulfhydryl groups in the receptor complex (e.g., see Bouzat et al, 1991;Gysin and Flanagan, 1987;Steinacker and Zuazaga, 1981;Walker et al, 1981;reviewed in Karlin, 2002). The inhibitory synapses mediated by the ionotropic GABA A -c-aminobutyric acid and glycine-gated channels are also regulated by the redox status of resident thiol groups.…”