Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins
Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. The effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. Results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. Results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits.
- Research Organization:
- Institute for Developmental Research, Aichi Prefectural Colony, Japan
- OSTI ID:
- 5736958
- Journal Information:
- Mol. Pharmacol.; (United States), Vol. 3
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
IMIDES
BIOLOGICAL EFFECTS
PROTEINS
RECEPTORS
BRAIN
BUTYRIC ACID
CATTLE
CELL MEMBRANES
IN VITRO
INHIBITION
MAGNESIUM
TRITIUM COMPOUNDS
ALKALINE EARTH METALS
ANIMALS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
DOMESTIC ANIMALS
ELEMENTS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
METALS
MONOCARBOXYLIC ACIDS
NERVOUS SYSTEM
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
RUMINANTS
VERTEBRATES
560301* - Chemicals Metabolism & Toxicology- Cells- (-1987)
550201 - Biochemistry- Tracer Techniques