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Characterisation of L‐[3H]glutamate binding to fresh and frozen crude plasma membranes isolated from cerebral cortex of adult rats
Author(s) -
Emanuelli T.,
Antunes V. F.,
Souza D. O. G.
Publication year - 1998
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216549800202362
Subject(s) - glutamate receptor , ampa receptor , membrane , incubation , quisqualic acid , kainic acid , binding site , chemistry , biochemistry , kainate receptor , receptor
Specific [3H]glutamate binding to fresh crude plasma membranes (CPMs) was compared with binding to frozen CPMs and the optimal conditions for the binding to frozen CPMs isolated from cerebral cortex of adult rats were determined. Freezing reduced [3H]glutamate binding (3.5‐fold), and pre‐incubation of previously frozen membranes followed by three washes increased binding (4.5‐fold) when compared to fresh samples. CPMs washed once, pre‐incubated at 37°C and washed 3 times was adopted as the most adequate condition for the binding assay of frozen membranes. In a Cl‐‐containing medium, [3H]glutamate binding (Bmax=97.9 pmol/mg, Kd=349.68 nM) to this frozen CPM preparation was significantly displaced by excess quisqualic acid (QA) (65%), L‐2‐amino‐4‐phosphonobutyric acid (L‐AP4) (35%), trans‐1‐aminocyclopentane‐1,3‐dicarboxylate (1S,3R‐ACPD) (25%) and alfa‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) (25%). In a Cl‐‐free medium, binding (Bmax=44.14 pmol/mg, 311 nM) was significantly displaced by QA (45%), L‐AP4 (25%), ACPD (25%), AMPA (25%), kainic acid (20%) and N‐methyl‐D‐aspartate (15%).