Characterization of L-[ 3H]glutamate binding sites in bovine brain coated vesicles

Abstract

Clathrin-coated vesicles isolated from bovine brain exhibit an L-[ 3H]glutamate-specific binding. Coated vesicles were purified from bovine brain by differential centrifugation and gel filtration. High purity of coated vesicles was established previously by several enzyme markers and electron microscopy. The binding activity was performed in the absence of Na +, Ca 2+, and Cl − ions to avoid binding and/or uptake to uptake sites. Coated vesicles were frozen, thawed, treated with 0.04% Triton X-100 and washed before incubation with L-[ 3H]glutamate. Saturation binding experiments revealed a single binding sitewith a K d = 439 ± 87 nM and a B max = 11.74 ± 3.4 pmol/mg protein, consistent with kinetics characteristic for glutamate receptors. The glutamate-specific binding was stereospecific for glutamate and aspartate, showing higher affinity for L-forms than D-forms. Pharmacological characterization indicated that specific binding was sensitive to quisqualate and almost insensitive to kainate and N-methyl-D-aspartate. 200 μM guanosine triphosphate (GTP) produced a decrease of 50% in L-[ 3H]glutamate binding activity and competition experiments produced an affinity shift to the right of the glutamate dose-response curve. These results support the evidence that glutamate receptors are present in bovine brain coated vesicles and, at least in part, are associated to a G-protein.