Solubilisation of a novel anticonvulsant binding site from pig cortical membranes.
J Recept Signal Transduct Res
; 20(2-3): 167-86, 2000.
Article
in En
| MEDLINE
| ID: mdl-10994652
ABSTRACT
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, [125I]-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. [125I]-SB-217644 was an ideal mechanistic tool for quantifying the binding to this novel anticonvulsant site, with a high specific activity and affinity (K(D) of 3 nmol/l). Optimum conditions for the solubilisation of this anticonvulsant binding site were investigated using a multifactorial experimental 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 yields of binding activity (25%) were achieved with a low concentration of the zwitterionic detergent, CHAPS, in the presence of a low protein concentration. This yield was further enhanced on combining mixtures of detergents. The highest recovery (37%) was achieved with a 5050 (vv; 1.5 x critical micelle concentration) mixture of the ionic detergent, sodium cholate, and the non-ionic detergent, MEGA-10. In summary, we report the successful solubilisation of a novel anticonvulsant binding site, identified by its selective affinity for SB-204269 and its analogues. The recovery of nearly 40% of the target binding sites from the starting material should provide a good starting point for the purification of this protein.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Benzamides
/
Benzopyrans
/
Cerebral Cortex
/
Anticonvulsants
Type of study:
Prognostic_studies
Limits:
Animals
Language:
En
Journal:
J Recept Signal Transduct Res
Journal subject:
BIOQUIMICA
/
FISIOLOGIA
Year:
2000
Document type:
Article