Microbial methodological artifacts in [3H]glutamate receptor binding assays.
Incubation of radiolabeled L-glutamic acid, a putative central excitatory neurotransmitter, in 50 mM Tris-acetate buffer (pH 7.4) at 30 degrees C in the absence of brain synaptic membranes resulted in a significant adsorption of the radioactivity to glass fiber filters routinely employed to trap the bound ligand in receptor binding assays. The adsorption was not only eliminated by the inclusion of L-isomers of structurally related amino acids, but also inhibited by that of most presumed agonists and antagonists for the brain glutamate receptors. This displaceable adsorption was a temperature-dependent nonreversible, and saturable phenomenon. Scatchard analysis of these data revealed that the adsorption consisted of a single component with an apparent dissociation constant of 73 nM. The displaceable adsorption was significantly attenuated by a concurrent incubation with papain, pronase E, and phospholipase C. A significant amount of the radioactivity was detected in the pass-through fraction of the Dowex column following an application of the reaction mixture incubated with purified [3H]glutamate at 30 degrees C for 60 min in the absence of membranous proteins added. Complete abolition of the displaceable adsorption resulted from the use of incubation buffer boiled at 100 degrees C as well as filtered through a nitrocellulose membrane filter with a pore size of 0.45 micron immediately before use. These results suggest that the displaceable adsorption may be attributable to the radioactive metabolite of [3H]glutamate by microorganisms contaminating the Tris-acetate buffer. This might in part contribute to some of the controversial results with regard to receptor binding studies on acidic amino acids.
Yoneda
Y
Y
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Ogita
K
K
D000327:Adsorption; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D011865:Radioisotope Dilution Technique; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*; D014316:Tritium
D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D014316:Tritium