Neurochem. Int. Vol. 11, No. 2, pp. 209-218, 1987 0197-0186/87 $3.00+ 0.00 Printed in Great Britain. All rights reserved Copyright © 1987 PergamonJournals Ltd CORRELATION OF GLUTAMATE BINDING ACTIVITY WITH GLUTAMATE-BINDING PROTEIN IMMUNOREACTIVITY IN THE BRAIN OF CONTROL AND ALCOHOL-TREATED RATS E. MICHAELIS, S. ROY, N. GALTON, M. CUNNINGHAM, E. LECLUYSE and M. MICHAELIS Neurobiology Section, Department of Human Development and Center for Biomedical Research, University of Kansas, Lawrence, KS 66045, U.S.A. (Received 9 May 1986; accepted 25 March 1987) Abstract--Specificantibodies raised against a glutamate binding protein purified from bovine brain were used to trace the immunoreactivity of this protein in rat brain subcellular fractions. In the subcellular fractions obtained from whole brain homogenates, the synaptic membranes had the highest immuno- chemical reactivity towards the anti-glutamate-binding protein antibodies. The combination of measure- ments of glutamate binding activity and glutamate-binding protein immunoreactivity indicated that in brain synaptic membranes from control animals the highest activity in these two measures was associated with a synaptic plasma membrane subfraction that was enriched with synaptic junctions. In animals treated with ethanol for 14 days, there was a significant increase in the density of synaptic membrane glutamate binding sites. This increase in glutamate binding capacity was correlated with a greater than two-fold increase in the glutamate binding activity and binding protein immunoreactivity of the light synaptic membrane subfraction, a subfraction which does not contain many recognizable synaptic junctions. Acute administration of ethanol to rats produced a moderate but non-significant decrease in glutamate binding capacity of synaptic membranes. The increase in the number of glutamate binding protein subunits in brain plasma membranes may be an adaptive response of central nervous system neurons to the acute effects of ethanol on glutamate synaptic transmission. L-Glutamate binding sites in isolated plasma mem- branes from the synaptic region of brain neurons have been studied by several investigators because they may represent the recognition sites of the phys- iologic receptors with which L-glutamic acid interacts to bring about neuronal excitation (Foster and Roberts, 1980; Bizierre et al., 1980; Foster et al., 1981; Michaelis et al., 1981; Baudry and Lynch, 1981; Werling and Nadler, 1982; Fagg and Foster, 1984). An important criterion of the involvement of these glutamate binding sites in the function of the physiologic receptors for the excitatory amino acid L-glutamic acid is the localization of these binding entities at the neuronal plasma membrane and especially the synaptic membranes. It was previously shown that the density of glutamate binding sites is highest in the synaptic plasma membrane fraction obtained from rat brain homogenates (Michaelis et al., 1974, 1981; Foster and Roberts, 1980; Foster et al., 1981). A glutamate-binding protein has been purified from rat brain synaptic membranes following solubilization of the membranes and affinity chro- matographic separation of the binding protein (Michaelis, 1975; Michaelis et al., 1983c, 1984; Dambinova and Besedin, 1982). Antibodies against this glutamate binding protein have been raised in rabbits and were shown to inhibit glutamate-induced Na ÷ flux into synaptic mem- branes without any effect on the glutamate transport carriers (Roy and Michaelis, 1984; Roy et al., 1985). We have previously reported that this glutamate binding protein does not have any glutamate metab- olizing activity and that the selectivity of the active site of the protein for various amino acids, amino acid analogs, and other agents is distinguishable from that of the high-affinity, Na÷-gradient dependent L-glutamate transport carriers (Michaelis, 1975; Mi- chaelis et al., 1981, 1982, 1983c). In the present study we have tried to correlate the distribution of glu- tamate binding activity in homogenates from various brain regions and in synaptosomal membrane sub- fractions to the presence of the antigenic sites of the 209