Muscarinic receptors in porcine caudate nucleus. II. Different effects of N-ethylmaleimide on [3H]cis-methyldioxolane binding to heat-labile (guanyl nucleotide-sensitive) sites and heat-stable (guanyl nucleotide- insensitive) sites

T Nukada, T Haga and A Ichiyama

Heat treatment of membranes from porcine caudate nucleus (50 degrees for 7 min) caused a marked decrease in [3H]cis-methyldioxolane [( 3H]CD) binding without affecting seriously the binding of [3H]3- quinuclidinyl benzilate [( 3H]QNB). Approximately 20% of the [3H]CD binding at 5 nM [3H]CD remained after the heat treatment. The remaining binding was not affected by 0.1 mM guanylyl-5'-imidodiphosphate (GppNHp) or by nickel or other cations at concentrations below 10 mM. Treatment of the membranes with trypsin (30 micrograms/mg of protein) at 20 degrees for 20 min also caused a marked decrease in [3H]CD binding without affecting seriously the binding of [3H]QNB. About 20% of the original [3H]CD binding remained in the presence of trypsin at a high concentration of protein (90 micrograms/mg). N-Ethylmaleimide (NEM) affected [3H]CD binding in two different ways: (a) preincubation of the membranes with NEM caused a marked reduction in heat- and GppNHp- sensitive [3H]CD binding, and (b) treatment with NEM caused an enhancement of heat-, GppNHp-, and trypsin-insensitive [3H]CD binding. Neither of the NEM effects required the coexistence of agonists. The concentration of NEM required for the first effect was 10 times lower than that for the second effect, indicating the existence of two NEM- binding sites with different affinities for NEM. The equilibrium dissociation constant (Kd) for [3H]CD after NEM treatment was 33 nM and was not affected by GppNHp, Ni2+, or heat treatment; the Kd was only 4 times higher than that (8 nM) without NEM treatment. These findings indicated the existence of two kinds of [3H]CD binding sites with high affinities for agonists: one is sensitive to guanyl nucleotide and is abolished by NEM and the other is induced by NEM and insensitive to guanyl nucleotide.

Volume 24, Issue 3, pp. 374-379, 11/01/1983
Copyright © 1983 by American Society for Pharmacology and Experimental Therapeutics