CHARACTERIZATION OF L-[H-3]GLUTAMATE BINDING TO FRESH AND FROZEN CRUDE PLASMA-MEMBRANES ISOLATED FROM CEREBRAL-CORTEX OF ADULT RATS

Specific [H-3]glutamate binding to fresh crude plasma membranes (CPMs) was compared with binding to frozen CPMs and the optimal conditions f or the binding to frozen CPMs isolated from cerebral cortex of adult r ats were determined. Freezing reduced [H-3]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. C PMs washed once, pre-incubated at 37 degrees C and washed 3 times was adopted as the most adequate condition for the binding assay of frozen membranes. In a Cl--containing medium, [H-3]glutamate binding (Bmax=9 7.9 pmol/mg, Kd=349.68 nM) to this frozen CPM preparation was signific antly displaced by excess quisqualic acid (QA) (65%), L-2-amino-4-phos phonobutyric acid (L-AP4) (35%), trans-1-aminocyclopentane-1 ,3-dicarb oxylate (1S,3R-ACPD) (25%) and lfa-amino-3-hydroxy-5-methyl-4-isoxazol epropionate (AMPA) (25%). Ln a CL--free medium, binding (Bmax=44.14 pm ol/mg, 311 nM) was significantly displaced by QA (45%), L-AP4 (25%), A CPD (25%),AMPA (25%), kainic acid (20%) and N-methyl-D-aspartate (15%) .