Methods for removing endogenous factors from CNS membrane preparations: differences in [3H]GABA binding parameters and developmental-related effects.

The present report describes a systematic study comparing and combining methods currently used for the removal of endogenous factors known to affect the interaction of GABA with its receptor. The effects of these methods were analyzed by performing [3H]GABA binding studies, and by measuring the amount of residual GABA left in the different membrane preparations. The effectiveness of these methods were also applied to different developmental stages. The results show that: 1) an exhaustive buffer washing procedure is necessary to accurately measure the maximal binding capacity (Bmax) of the low-affinity GABA binding site, and 2) the use of more drastic methods, including freeze-thawing and Triton treatment allows a clear demonstration of receptor heterogeneity and a precise measurement of the Bmax of the high-affinity GABA binding site as well as increases the affinity of the low-affinity site. The analysis of the Bmax values obtained with these different procedures in relation to the values of GABA removal, strongly indicates that the exhaustive washing procedure removes some unknown endogenous substances required for Triton treatment to exhibit its maximal effectiveness. Finally, a detailed analysis of Kd and Bmax values obtained with these three methods in the developing nervous tissue shows the existence of significant differences with regard to their effectiveness in removing endogenous substances when applied in different developmental stages.