Solubilization and purification of a putative quisqualate-sensitive glutamate receptor from crustacean muscle.

Article date: 1991/1/1

PubMed ID: 2569700

Journal name: The Biochemical journal (ISSN: 0264-6021)


Two high-affinity glutamate-binding proteins have been solubilized and purified from crustacean muscle membranes. L-[3H]Glutamate binding to intact membranes gave IC50 values (concn. giving 50% inhibition) of 0.35 microM for glutamate. 9.0 microM for quisqualate and 36.2 microM for ibotenic acid. Kainate, domoate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, D-glutamate and L-aspartate were poor inhibitors of glutamate binding. The two protein were purified approx. 600-fold from muscle membranes by use of concanavalin A and gel-filtration column chromatography. One protein showed an apparent Stokes radius of 5.4 nm, as determined by gel filtration, with a subunit of Mr 74000 as shown by SDS/polyacrylamide-gel electrophoresis. Analysis of glutamate binding to this protein revealed a KD of 2.4 microM and a Bmax, of 15.5 nmol/mg, and a k1 for quisqualate inhibition of glutamate binding of 1.0 microM. The second protein was eluted anomalously from the gel-filtration column and showed a subunit of Mr 65000 on SDS/polyacrylamide gels. Glutamate binding to this protein showed a KD of 1.3 microM and a Bmax, of 14.5 nmol/mg, and was only slightly sensitive to the presence of quisqualate (Ki = 0.8 mM). Glutamate binding to both proteins was insensitive to kainate. It is concluded that the Mr-74000 protein is a prime candidate for the quisqualate-sensitive receptor of the crustacean neuromuscular junction.

Author List: Gray S R, Batstone F R, Santiapillai N F, Richardson P J

Publication Types: Journal Article; Research Support, Non-U.S. Gov't

Substances mentioned in the article: Glutamates; Receptors, Glutamate; Receptors, Neurotransmitter; Glutamic Acid; Quisqualic Acid;

Mesh terms: Animals; Binding Sites; Chromatography, Gel; Crustacea; Electrophoresis, Polyacrylamide Gel; Glutamates/metabolism; Glutamic Acid; Muscles/metabolism; Quisqualic Acid/pharmacology; Receptors, Glutamate; Receptors, Neurotransmitter/drug effects;

Citations: - 942051

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