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

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.

Abstract

Abstract: Specific L‐[ 3 H]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 [ 3 H]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 [ 3 H]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.