Problems Associated with the Binding of L-Glutamic Acid to Synaptic Membranes: Methodological Aspects

Abstract: Specific L-³H]glutamate binding was investigated in extensively washed synaptic membrane preparations from rat brain. Mild conditions of ultrasonication effected a significant enhancement of binding, attributable to the marked reduction in membrane vesicle size and the removal of endogenous interfering substances such as glutamate. Preincubation of freshly prepared membranes at 37°C for 30 min followed by further washing resulted in enhanced binding. Addition of supernatant from preincubated membranes effectively inhibited ³H]glutamate binding to control membranes; the possibility of the presence of an endogenous glutamate receptor inhibitor is discussed. Treatment of membranes with low concentrations of Triton X-100, in contrast with the findings for GABA, did not produce any significant enhancement of specific glutamate binding. While binding of ³H]glutamate is almost abolished in frozen or cold-stored membranes, lyophilisation had a remarkable effect, not only affording protection, but actually enhancing the binding properties of the synaptic membrane preparation.