Solubilisation of a novel anticonvulsant binding site from pig cortical membranes

The present study describes the solubilisation of the novel anticonvulsant, SB-204269, binding site from pig cortical membranes. Throughout the study the binding of a close analogue of this compound, [I-125]-SB-217644 (trans 6- Acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran -3R-ol) was used to monitor the success of the solubilisation procedure. [I -125]-SB-217644 was an ideal mechanistic tool for quantifying the binding t o this novel anticonvulsant site, with a high specific activity and affinit y (K-D of 3 nmol/l). Optimum conditions for the solubilisation of this anti convulsant binding site were investigated using a multifactorial experiment al design to assess a large number of variables. Detergent type, detergent- protein ratio, absence of Mg2+ and temperature were deemed to be important factors. However, the increases observed in binding site specific activity were minimal compared with those achieved for yields. Maximum percentage yi elds of binding activity (25%) were achieved with a low concentration of th e zwitterionic detergent, CHAPS, in the presence of a low protein concentra tion. This yield was further enhanced on combining mixtures of detergents. The highest recovery (37%) was achieved with a 50:50 (v:v; 1.5 x critical m icelle concentration) mixture of the ionic detergent, sodium cholate, and t he non-ionic detergent, MEGA-10. In summary, we report the successful solub ilisation of a novel anticonvulsant binding site, identified by its selecti ve affinity for SE-204269 and its analogues. The recovery of nearly 40% of the target binding sites from the starting material should provide a good s tarting point for the purification of this protein.