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

Michaelis, Elias K.; Michaelis, Mary L.; Stormann, Thomas M.; Chittenden, W. L.; Grubbs, Robert D.

doi:10.1111/j.1471-4159.1983.tb08150.x

Cited by 49 publications

(43 citation statements)

References 51 publications

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“…1B (12). Binding of a ligand to a self-associating acceptor macromolecule has been observed by others to give the appearance of the presence of multiple ligand binding sites (20,21).…”

Section: Resultsmentioning

confidence: 99%

“…[6][7][8][9][10]. In our previous studies we have concentrated on characterizing both the glutamate-binding sites and the glutamate-activated Na+ channels in mammalian brain synaptic membranes (11)(12)(13)(14)(15). We have also explored the biochemical features and binding activity and selectivity of a glutamate-binding glycoprotein purified from brain synaptic membranes (11)(12)(13).…”

mentioning

confidence: 99%

“…In our previous studies we have concentrated on characterizing both the glutamate-binding sites and the glutamate-activated Na+ channels in mammalian brain synaptic membranes (11)(12)(13)(14)(15). We have also explored the biochemical features and binding activity and selectivity of a glutamate-binding glycoprotein purified from brain synaptic membranes (11)(12)(13). The binding activity of this purified protein and its sensitivity to various chemical treatments are very similar to the activity and sensitivity of the high-affinity L-glutamate-binding sites in synaptic membranes (11)(12)(13).…”

mentioning

confidence: 99%

“…We have also explored the biochemical features and binding activity and selectivity of a glutamate-binding glycoprotein purified from brain synaptic membranes (11)(12)(13). The binding activity of this purified protein and its sensitivity to various chemical treatments are very similar to the activity and sensitivity of the high-affinity L-glutamate-binding sites in synaptic membranes (11)(12)(13).…”

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confidence: 99%

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Spider venoms inhibit L-glutamate binding to brain synaptic membrane receptors.

Michaelis¹,

Galton²,

Early³

1984

Proc. Natl. Acad. Sci. U.S.A.

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The venoms from three spider species, Araneus gemma, Neoscona arabesca, and Argiope aurantia, were shown to inhibit the high-affinity, sodium-independent L-glutamate-binding sites in rat brain synaptic membranes. The same three venoms caused concentration-dependent inhibition of the activity of the glutamate-binding glycoprotein purified from rat brain synaptic membranes. The venom milked from the glands of Araneus gemma was the most active inhibitor of L-glutamate binding, causing 60-80% inhibition of both synaptic membrane and purified protein binding activity at 0.01 unit. The inhibitory activity of this venom was associated with a single protein peak obtained from gel permeation chromatography of the venom. Finally, the effect of the venom from Araneus gemma on the synaptic membrane glutamate-binding sites was slowly reversible. These observations indicate that the spider venoms have a direct effect on the recognition sites for L-glutamic acid in brain synaptic membranes and that these sites are related to the physiologic glutamate receptors.The characterization of specific receptor sites with which Lglutamic acid interacts to produce excitation in vertebrate and invertebrate central nervous system neurons and in invertebrate neuromuscular junctions has been impeded by the lack of selective and potent antagonists of the excitatory activity of glutamate. A better definition of glutamate receptor sites in neurons may soon be possible due to recent observations by of the presence in spider venoms of selective antagonists of the L-glutamate excitatory activity detected in invertebrate neuromuscular junction and mammalian brain neuronal synapses. In the studies reported by Kawai et al. (1-3) the venom from two species of spiders, Nephila clavata and Araneus ventricosus, produced nearly identical inhibition of both neuronal pathway and L-glutamate-induced excitation at the lobster neuromuscular junction (1-3), a synaptic junction at which glutamate is thought to be the excitatory transmitter (4, 5).Several research groups have recently attempted to correlate the binding activity and selectivity of L-[3H]glutamatebinding sites in neuronal membranes with the characteristics of the physiologic L-glutamate receptors (e.g., refs. 6-10). In our previous studies we have concentrated on characterizing both the glutamate-binding sites and the glutamate-activated Na+ channels in mammalian brain synaptic membranes (11-15). We have also explored the biochemical features and binding activity and selectivity of a glutamate-binding glycoprotein purified from brain synaptic membranes (11-13). The binding activity of this purified protein and its sensitivity to various chemical treatments are very similar to the activity and sensitivity of the high-affinity L-glutamate-binding sites in synaptic membranes (11)(12)(13) MATERIALS AND METHODS Preparation of Synaptic Membranes and Measurement of L-[3H]Glutamate Binding. The synaptic plasma membranes used in these studies were purified from rat (male, SpragueDawley, Charles Ri...

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“…1B (12). Binding of a ligand to a self-associating acceptor macromolecule has been observed by others to give the appearance of the presence of multiple ligand binding sites (20,21).…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Spider venoms inhibit L-glutamate binding to brain synaptic membrane receptors.

Michaelis¹,

Galton²,

Early³

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Prior to the identification of NMDAR subunit cDNAs and antibodies, isolation of NMDAR had been attempted using NMDAR ligand affinity methods (Michaelis et al . 1992, 1983).…”

Section: Resultsmentioning

confidence: 99%

Isolation of 2000‐kDa complexes of N‐methyl‐d‐aspartate receptor and postsynaptic density 95 from mouse brain

Husi

Grant

2001

Journal of Neurochemistry

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Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR±PSD-95 complexes from mouse brain including immunoaf®nity, immunoprecipitation, ligand-af®nity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel ®ltration indicated the native NMDAR±PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to de®ne the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery. Keywords: hebbosome, immunoaf®nity, NMDA receptor, peptide af®nity, postsynaptic density 95, signalling complexes.Large multiprotein complexes linking receptors to intracellular proteins are emerging as a general mechanism for conducting a variety of cellular functions. The synapse also appears to contain a number of important presynaptic and postsynaptic complexes regulating synaptic transmission and synaptic plasticity. At the presynaptic terminal, the traf®cking, docking and exocytosis of synaptic vesicles involves complexes of proteins including snare complexes (reviewed by Catterall 1999). On the postsynaptic side, most neurotransmitter receptors and channels are now known to bind to intracellular proteins that are involved with signal transduction. A neurotransmitter receptor, for which a vast body of pharmacological literature demonstrates its importance in the physiology of stroke, pain and other conditions, is the N-methyl-d-aspartate receptor (NMDAR). Despite the importance of this receptor, there have been no reports of general methods for its physical isolation as a large complex with its associated proteins from intact brain. These methods are essential for characterization of these in vivo complexes and will provide a basis for proteomic characterization, structural studies as well as new approaches to drug screening for agents modifying the pathological conditions involving the NMDAR.The ®rst described NMDAR interacting protein was postsynaptic density 95 (PSD-95) (Kornau et al. 1995), an intracellular protein found in postsynaptic density (PSD) preparations. The physiological role of PSD-95 was revealed by mouse gene targeting experiments where it was shown to regulate the coupling of the NMDAR to downstream pathways that regulate ...

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Immunocytochemistry of neurotransmitter receptors

Wenthold

Altschuler

Hampson

1990

J. Elec. Microsc. Tech.

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Over the last several years our knowledge of neurotransmitter receptors has increased dramatically as receptor types and subtypes have been identified through the development of selective antagonists, neuropharmacological studies, and radioactive ligand binding studies. At the same time major advances were made in the immunocytochemical localization of neurotransmitters and their related enzymes. However, only recently has immunocytochemistry been used to localize neurotransmitter receptors, and these studies have been limited. Four receptors have been localized in the CNS with immunocytochemistry: the nicotinic acetylcholine receptor, the beta-adrenergic receptor, the GABA/benzodiazepine receptor, and the glycine receptor. Of these the glycine receptor has been the most thoroughly characterized. Glycine receptor immunoreactivity is highly concentrated at postsynaptic sites, and the distribution of immunoreactivity appears to correlate closely with glycinergic neurons. However, immunocytochemical studies done on other receptors suggest such a distribution may not always be the case. Some receptors may not be concentrated at postsynaptic sites, and receptor distribution may not always closely fit the distribution of the respective neurotransmitter. Work is rapidly progressing on the purification of other receptors and on the production of selective antibodies which will allow immunocytochemical studies which address these and other questions.

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

Cited by 49 publications

References 51 publications

Spider venoms inhibit L-glutamate binding to brain synaptic membrane receptors.

Spider venoms inhibit L-glutamate binding to brain synaptic membrane receptors.

Isolation of 2000‐kDa complexes of N‐methyl‐d‐aspartate receptor and postsynaptic density 95 from mouse brain

Immunocytochemistry of neurotransmitter receptors

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