MUSCARINIC RECEPTORS IN PORCINE CAUDATE-NUCLEUS .2. DIFFERENT EFFECTS OF N-ETHYLMALEIMIDE ON [H-3]CIS-METHYLDIOXOLANE BINDING TO HEAT-LABILE (GUANYL NUCLEOTIDE-SENSITIVE) SITES AND HEAT-STABLE (GUANYL NUCLEOTIDE-INSENSITIVE) SITES

  • 1 January 1983
    • journal article
    • research article
    • Vol. 24  (3) , 374-379
Abstract
Heat treatment of membranes from porcine caudate nucleus (50.degree. C 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). Of the [3H]CD binding at 5 mM [3H]CD .apprx. 20% remained after the heat treatment. The remaining binding was not affected by 0.1 mM guanylyl-5''-imidodiphosphate (GppNHp) or by Ni2+ or other cations at concentrations below 10 mM. Treatment of the membranes with trypsin (30 .mu.g/mg of protein) at 20.degree. C for 20 min also caused a marked decrease in [3H]CD binding without affecting seriously the binding of [3H]QNB. Of the original [3H]CD binding .apprx. 20% remained in the presence of trypsin at a high concentration of protein (90 .mu.g/mg). N-Ethylmaleimide (NEM) affected [3H]CD binding in 2 different ways: preincubation of the membranes with NEM caused a marked reduction in heat- and GppNHp-sensitive [3H]CD binding, and 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 1st effect was 10 times lower than that for the 2nd effect, indicating the existence of 2 NEM-binding sites with different affinities for NEM. 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 n) without NEM treatment. Apparently there are 2 kinds of [3h]CD binding sites with high affinities for agonists: 1 is sensitive to guanyl nucleotide and is abolished by NEM and the other is induced by NEM and insensitive to guanyl nucleotide.

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