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SOLUBILIZATION AND PARTIAL PURIFICATION OF THE GABA RECEPTOR FROM MOUSE BRAIN AND A BINDING ASSAY FOR THE SOLUBILIZED RECEPTOR1

Authors

  • Obi Chude

    1. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90024, U.S.A.
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    • 2

      Department of Medical Biochemistry, University of Nigeria, Enugu Campus, Enugu, Anambra State, Nigeria. All reprint requests should be sent to this address.


  • 1

    This work was supported by Grant No. PCM75-1884 of the National Science Foundation, Paul D. Boyer, Principal Investigator.

Abstract

Abstract— The GABA receptor from mouse brain was solubilized with lysolecithin. A 56-fold overall purification and activation were achieved by discontinuous sucrose gradient centrifugation and solubilization. Activation of binding by both procedures was observed. The solubilized receptor has the following binding constants: KD1= 3.5 nM, KD2= 52 nM, Bmax 1= 2.8 pmol/mg protein and Bmax 2= 14 pmol/mg protein for muscimol; KD1= 12 nM, KD2= 470 nM, Bmax 1= 1.4 pmol/mg protein and Bmax 2= 17 pmol/mg protein for GABA. Specific GABA binding was inhibited by imidazoleacetic acid and bicuculline with IC50 values of 250nM and 1 μM respectively. A rapid and sensitive filtration binding assay for the solubilized receptor has been developed. Lysolecithin was also found suitable for the solubilization of acetylcholine receptor from T. californica electroplaques.

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