Purification and functional analysis of Ric-8A: a guanine nucleotide exchange factor for G-protein alpha subunits

Methods Enzymol. 2004;390:377-88. doi: 10.1016/S0076-6879(04)90023-7.

Abstract

Ric-8A (synembryn) has been shown to accelerate the in vitro guanine nucleotide exchange activities of most G-protein alpha subunits (with the exception of Galphas). Methods are presented in this article for the purification of Ric-8A and functional analysis of the effects Ric-8A has on G-protein alpha subunit guanine nucleotide-binding activities. The use of Ric-8A to prepare GTPgammaS-Galpha and nucleotide-free Galpha (in complex with Ric-8A) is described.

MeSH terms

  • Animals
  • Chromatography / methods
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Protein alpha Subunits / metabolism*
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / isolation & purification*
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / isolation & purification
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Guanosine Triphosphate / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / isolation & purification*
  • Nuclear Proteins / metabolism*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction / physiology

Substances

  • GTP-Binding Protein alpha Subunits
  • Guanine Nucleotide Exchange Factors
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Ric8a protein, rat
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Guanosine Triphosphate
  • GTP Phosphohydrolases