Solubilization of quisqualate-sensitive [3H]glutamate binding activity from rat retina.
Binding activity of a putative central neurotransmitter, L-glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P-40. [3H]Glutamate binding activity 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 increasing with incubation time up to 60 min at 30 degrees C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30 degrees C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 microM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to L-glutamate, and was abolished by quisqualate, L-glutamate diethyl ester, and DL-2-amino-3-phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation-exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30 degrees C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate-sensitive membranous enzyme with a stereospecific and structure-selective high affinity for the central neurotransmitter.
Yoneda
Y
Y
Department of Pharmacology, Setsunan University, Hirakata, Osaka, Japan.
Ogita
K
K
D015763:2-Amino-5-phosphonovalerate / Q000031:analogs & derivatives*; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism* / Q000494:pharmacology; D018698:Glutamic Acid; D007700:Kinetics; D008297:Male; D016202:N-Methylaspartate; D017830:Octoxynol; D010069:Oxadiazoles / Q000494:pharmacology*; D011092:Polyethylene Glycols; D016318:Quisqualic Acid; D051381:Rats; D011919:Rats, Inbred Strains; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D012160:Retina / Q000187:drug effects / Q000378:metabolism*; D012995:Solubility
D000596:Amino Acids; D005971:Glutamates; D010069:Oxadiazoles; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; C011829:glutamic acid diethyl ester; D001224:Aspartic Acid; D018698:Glutamic Acid; D011092:Polyethylene Glycols; D016202:N-Methylaspartate; D015763:2-Amino-5-phosphonovalerate; D016318:Quisqualic Acid; D017830:Octoxynol; C010615:Nonidet P-40; C031231:2-amino-7-phosphonoheptanoic acid