Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1984

OSTI ID:7024611

Klotz, K L

In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

• Research Organization: Georgetown Univ., Washington, DC (USA)
• OSTI ID: 7024611
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
AMINOBUTYRIC ACID
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
AZINES
BIOCHEMISTRY
BODY
BRAIN
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
CHEMICAL REACTIONS
CHEMISTRY
CHROMATOGRAPHY
DECOMPOSITION
DRUGS
ELECTROPHORESIS
ENZYMATIC HYDROLYSIS
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROLYSIS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
LYSIS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PEPTIDE HYDROLASES
PROTEINS
RATS
RECEPTORS
RODENTS
SEPARATION PROCESSES
SERINE PROTEINASES
SOLVOLYSIS
TRITIUM COMPOUNDS
TRYPSIN
VERTEBRATES