High affinity glutamate binding during postnatal development of rat cerebellum

127

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.

Section: Discussionsupporting

confidence: 91%

Characterization of Two [³H]Glutamate Binding Sites in Rat Hippocampal Membranes

Baudry

Lynch

1981

127

“…The physiological effects of L-glutamic acid on central nervous system neurons are produced through interaction with membrane receptors that face towards the extracellular environment (Coombs et al, 1955). The neuronal membrane receptors for L-glutamic acid have been explored through studies of the binding interactions of ~-["H]glutamate with particulate and isolated membrane preparations (e.g., Michaelis et a]., 1974; Foster and Roberts, 1978;Bizierre et al, 1980;de Barry et al, 1980;Baudry and Lynch, 1981;Foster et al, 1981).…”

mentioning

confidence: 99%

Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate‐Binding Protein

Michaelis

Stormann

et al. 1983

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.

“…The identification and characterization of the physiological glutamic acid (GLU)-receptors in the mammalian central nervous system using ligand binding techniques would be of great importance for the understanding of the neurotransmitter function of GLU. Several attempts have been made to use [3H]GLU as a ligand for detection of GLU receptors in vitro (Michaelis et al, 1974;Roberts, 1974;Michaelis, 1975;De Robertis and Fiszer de Plazas, 1976;Baudry and Lynch, 1979;de Barry et al, 1980;Biziere et al, 1980), but the characteristics of the binding sites so far detected are quite different and these sites might not be directly related to the physiological GLU receptors. Foster and Roberts (1978) have demonstrated [3H]GLU binding sites on fresh cerebellar membranes from rat brain.…”

mentioning

confidence: 99%

Ibotenic Acid Analogues as Inhibitors of [³H]Glutamic Acid Binding to Cerebellar Membranes

Honoré

Lauridsen

Krogsgaard‐Larsen

1981

The L-[3H]glutamic acid binding capability of rat cerebellar membranes prepared with or without preincubation at 37 degrees C followed by washing was investigated. The two preparations (KD = 820 nM, Bmax = 54.5 pmol/mg protein; KD = 509 nM, Bmax = 13.0 pmol/mg protein) showed no difference in specificity of the binding of the ibotenic acid analogues, consistent with the removal of an endogenous inhibitor by the preincubation at 37 degrees C followed by washing. The order of potency of the ibotenic acid analogues as inhibitors of L-[3H]glutamic acid binding is different from the order of potency in vivo, suggesting that the binding sites found are different from the physiological glutamic acid receptor.