Reassessment of [3H]glutamate binding to human brain membrane preparations

We studied [3H]glutamate binding to human brain membrane preparations. We found that the binding assay of [3H]glutamate should be done at 4 degrees C using glass-fiber filters pretreated with polyethyleneimine, sterile incubation buffer, and membrane preparations pretreated with a detergent. Under these assay conditions, we have characterized the three subtypes of glutamate receptors: N-methyl-D-aspartate-, quisqualate- and kainate-sensitive binding sites. Scatchard analysis revealed that the N-methyl-D-aspartate-sensitive [3H]glutamate binding sites consisted of a single component with a dissociation constant (Kd) of 28.2 +/- 2.8 nM and total concentration (Bmax) of 36.2 +/- 3.6 fmol/mg protein (N = 8). These data were much different from those presented in previous studies.