Solubilization and purification of a putative quisqualate-sensitive glutamate receptor from crustacean muscle.
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.
Gray
S R
SR
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Batstone
F R
FR
Santiapillai
N F
NF
Richardson
P J
PJ
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003445:Crustacea; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D009132:Muscles / Q000378:metabolism*; D016318:Quisqualic Acid / Q000494:pharmacology*; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification*
D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; D018698:Glutamic Acid; D016318:Quisqualic Acid