The γ-aminobutyric-acid/benzodiazepine-receptor protein from rat brain

Large-scale purification and preparation of antibodies

Authors

  • Gregory B. STAUBER,

    1. Department of Biochemistry, University of California, Riverside
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  • Richard W. RANSOM,

    1. Department of Pharmacology, School of Medicine and Brain Research Institute, University of California, Los Angeles
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  • Aydin I. DILBER,

    1. Department of Pharmacology, School of Medicine and Brain Research Institute, University of California, Los Angeles
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  • Richard W. OLSEN

    Corresponding author
    1. Department of Biochemistry, University of California, Riverside
    2. Department of Pharmacology, School of Medicine and Brain Research Institute, University of California, Los Angeles
      Correspondence to R. W. Olsen, Department of Pharmacology, Center for the Health Sciences 23–278, UCLA School of Medicine, Los Angeles, California, USA-90024
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Correspondence to R. W. Olsen, Department of Pharmacology, Center for the Health Sciences 23–278, UCLA School of Medicine, Los Angeles, California, USA-90024

Abstract

The γ-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound γ-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168–171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble γ-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.

Abbreviation
GABA

γ-aminobutyric acid

Ancillary

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