Possible existence of loosely membrane-bound specific binding sites for phenytoin.

Abstract

In recent years, specific high-affinity sites for potent drugs such as morphine and diazepam have been demonstrated in the brain. For phenytoin, one of the most commonly used drugs for epilepsy, the existence of the specific binding sites was proposed in our previous report: on the basis of studies using the ammonium sulfate precipitationglass filter filtration method. Burnham et al.l have also reported such specific binding sites recently, whereas other authors2 have failed to find the specific binding activity for phenytoin. In the present study, it was observed that the solubilized binding sites from the particulate fraction had a dissociation constant (Kd) value similar to that of the supernatant fraction. This suggests the existence of the sites which are loosely bound to the membranes. tained by means of the second centrifugation, consisted of crude mitochondria1 and microsoma1 fractions and were designated the particulate fraction. For solubilization, the particulate fraction was frozen at 8OoC for about 30 min, thawed, ultrasonicated and centrifuged at 200,000 X g for 60 min. The solubilized fraction was dialyzed against a 50mM Tris-HCI buffer (pH7.5) and concentrated. To measure the regional distribution of the binding activity in the rat brain, both the solubilized and supernatant fractions were prepared together due to the small amount of materials. The 800 X g supernatant was directly frozen and solubilized as described above. The assay for the specific binding was carried out with slightly modified versions of the method described in the previous report,I as shown in the legend of Fig. 1.