Jones S M, Snell L D, Johnson K M
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77550.
J Pharmacol Methods. 1989 Apr;21(2):161-8. doi: 10.1016/0160-5402(89)90034-x.
Rapid vacuum filtration assays were used to quantitate radioligand binding to phencyclidine (PCP), N-methyl-D-aspartate (NMDA), and strychnine-insensitive glycine receptors in a simple buffy coat preparation of rat cortical membranes. KD and Bmax values for [3H]glycine binding were very similar to those previously reported by workers who used centrifugation for the separation of free and bound [3H]glycine. We also found that this preparation had a high percentage of NMDA-displaceable L-[3H]glutamate binding sites, which demonstrated a pharmacology very similar to that previously observed in more purified synaptic plasma membranes. Hill analysis of the displacement curves indicated that glutamate bound to a single class of sites, but that NMDA and NMDA antagonists may interact with this site in a negatively cooperative fashion. This preparation was also found to be suitable for the study of NMDA and glycine receptor regulation of the associated ion channel, as these effectors, alone and in combination, increased the affinity with which [3H]TCP bound to the PCP receptor believed to be located within the ion channel. Thus, the ability to measure radioligand binding to these three sites in the same simple membrane preparation should greatly facilitate the study of the interaction between them.
