Characterization of a cytosolic protein (P36) isolated from pig brain by benzodiazepine-affinity chromatography.
Benzodiazepine-affinity chromatography, on a column of 1012S-Sepharose, resulted in the detection and purification of a binding protein (P36) from the cytosolic fraction of pig cerebral cortex. Purified P36 was enriched over 3,500-fold in a single step and was recovered with an efficiency of 50-60%. Analysis of the purified preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis demonstrated a single polypeptide of Mr 36,000. The Stokes radius (3.44 nm) and sedimentation coefficient (4.43S) indicated that purified P36 is a dimeric protein. Analysis of the amino acid composition of P36 revealed a relatively high content of the hydrophobic amino acids, valine and leucine. Immunoblotting of several pig tissue preparations with an antiserum raised against purified P36 demonstrated approximately equal enrichment of P36 in cerebral cortex, cerebellum, and adrenal glands. Lesser enrichment was observed in kidney and liver, whereas a number of other tissues displayed no immunoreactivity. The gamma-aminobutyrate/benzodiazepine receptor complex and P36 showed no immunological cross-reactivity. High-affinity binding activity for [3H]Ro 15-4513, [3H]flunitrazepam, or [3H]PK11195 was not detected in preparations of purified P36. However, the ability of the gamma-aminobutyrate/benzodiazepine receptor inverse agonists, methyl- and ethyl-beta-carboline-3-carboxylate, to inhibit the binding of P36 to 1012S-Sepharose at relatively low concentrations indicates that P36 exhibits a degree of binding specificity.
Kirkness
E F
EF
Department of Biochemistry, University of Leeds, England.
Turner
A J
AJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism*; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D008970:Molecular Weight; D013552:Swine; D014018:Tissue Distribution
D000596:Amino Acids; D002352:Carrier Proteins; C054568:benzodiazepine-binding protein p36; D001569:Benzodiazepines