L‐[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

: The binding of L‐[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L‐[3H]glutamate. The preparation showed high specific binding of L‐glutamate with a KD of 0.12 μM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4°C. Nonspecific binding was 5–10% of total binding. The gluta‐mate binding was highly stereospecific [K0.5 (D‐glutamate), 270 μM] and showed a high degree of discrimination between l‐glutamate and l‐aspartate [K0.5 (L‐aspartate), 54 μM]. In mammalian CNS preparations potent agonists of l‐glutamate such as kainate and N‐methyl‐d‐aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of l‐glutamate binding [K0.5 (quisqualate), 162 μM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 μM], and various esters of l‐glutamate were of intermediate potency as displacers of l‐[3H]glutamate binding (K0.5 values from 6 to 60 μM) The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra‐junctional or a junctional receptor.