Purification and characterization of a protein associated with peripheral-type benzodiazepine binding sites.
The photoaffinity ligand [3H]PK 14105 was utilized to modify covalently peripheral-type benzodiazepine binding sites in rat adrenal mitochondrial preparations. The photolabeled membrane preparations were then solubilized in 1% digitonin and the detergent-soluble extracts subjected to fractionation by ion-exchange chromatography and reversed-phase high performance liquid chromatography. This scheme resulted in the purification of the putative binding site protein for PK 14105 which we have entitled PKBS. Purified preparations of PKBS exhibited a single band with a Mr of approximately 17,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver-staining or autoradiographic detection. Additional criteria examining the purity of PKBS preparations were provided by radioiodination with Bolton-Hunter reagent, amino acid analysis, gas-phase sequencing, and reversed-phase chromatography suggesting that this protein was purified to apparent homogeneity. These results demonstrate that a protein associated with peripheral-type benzodiazepine recognition sites has been isolated thus facilitating more direct studies on the structure of this receptor and on the role of these binding sites in mediating responses elicited by benzodiazepines acting at these sites.
Antkiewicz-Michaluk
L
L
FIDIA-Georgetown Institute for the Neurosciences, Georgetown University Medical School, Washington, D.C. 20007.
Mukhin
A G
AG
Guidotti
A
A
Krueger
K E
KE
D000311:Adrenal Glands / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007546:Isoquinolines / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011963:Receptors, GABA-A / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D000596:Amino Acids; D007546:Isoquinolines; D011963:Receptors, GABA-A; C051238:PK 14105