Solubilization of stereospecific and quisqualate-sensitive activity of [3H]glutamate binding in the pituitary of the rat.
Binding activity of a putative central excitatory neurotransmitter, L-glutamic acid, was solubilized from the pituitary glands of the rat by treatment of the membranous homogenates with a nonionic detergent, Nonidet P-40. The binding activity of [3H]glutamic acid increased linearly with increasing concentrations of the solubilized proteins, up to 15 micrograms. The binding activity reached an equilibrium within 10 min at 2 degrees C, while the time required to attain equilibrium at 30 degrees C was 60 min. Addition of an excess of nonradioactive glutamic acid rapidly decreased the activity detected at 30 degrees C, to the nonspecific binding level. Scatchard analysis of these data revealed that the solubilized binding activity consisted of a single component with a Kd of 0.34 microM and a Bmax of 53.6 pmol/mg protein. L-Glutamic but not D-glutamic acid inhibited the binding activity in a concentration-dependent manner, at the concentration range greater than 10(-8) M. An agonist for a certain subclass of the central glutamate receptors, quisqualic acid, significantly inhibited the solubilized activity, whereas the other two agonists, such as N-methyl-D-aspartic acid and kainic acid, had no significant effect. Reduction of the incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to a decrement in the number of apparent binding sites. These results suggest that the binding activity of [3H]glutamic acid in the pituitary may be derived from a quisqualate-sensitive membranous constituent with a stereospecific high affinity for the central neurotransmitter.
Yoneda
Y
Y
Department of Pharmacology, Setsunan University, Osaka, Japan.
Ogita
K
K
D000818:Animals; D002852:Chromatography, Ion Exchange; D005971:Glutamates / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009467:Neuromuscular Depolarizing Agents / Q000494:pharmacology*; D010069:Oxadiazoles / Q000494:pharmacology*; D010902:Pituitary Gland / Q000378:metabolism*; D016318:Quisqualic Acid; D051381:Rats; D011919:Rats, Inbred Strains; D013237:Stereoisomerism
D005971:Glutamates; D009467:Neuromuscular Depolarizing Agents; D010069:Oxadiazoles; D016318:Quisqualic Acid