Effects of pentobarbital on t-[35S]butylbicyclophosphorothionate and [3H]flunitrazepam binding to membrane-bound and solubilized preparations from rat forebrain

Abstract

The binding sites for t-[35S]butylbicyclophosphorothionate ([35S] TBPS) and [3H]flunitrazepam ([3H]Flu) were solubilized from freshly prepared and washed membranes from rat forebrain with 20 mM 3-[(cholamidopropyl)-dimethylamonio]-1-propanesulfonate in the presence of protease inhibitors. Approximately 64% of the protein, 56% of the [35S]TBPS sites and 45% of the [3H] Flu sites were solubilized. The soluble and membrane-bound binding sites for [3H]Flu were relatively stable on storage at 0 degree C or at -65 degrees C for 11 days. Binding of [35S]TBPS to membranes was increased after freezing the membranes at -65 degrees C for 1 to 11 days and was relatively unchanged if the membranes were stored at 0 degree C for 1 to 11 days. Solubilized [35S]TBPS binding sites were stable when stored at -65 degrees C but declined to 20% of control when stored at 0 degree C for 11 days. Tested shortly after preparation, [35S]TBPS binding to both soluble and membrane preparations demonstrated similar affinity, temperature dependence and noncompetitive inhibition by pentobarbital (pb). Stimulation by pb of [3H]Flu binding to the soluble fraction decayed on storage of the solubilized material at 0 degree C with a time course similar to the loss of [35S]TBPS binding. This finding might suggest that the stimulation of [3H]Flu binding by pb is mediated via its action on TBPS sites. However in membrane preparations stored at 0 degree C, [35S]TBPS binding was stable whereas the pb effects declined with time.(ABSTRACT TRUNCATED AT 250 WORDS)