32] Isolation, cloning, and characterization of brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factor

Abstract

This chapter describes the isolation, cloning, and characterization of Brefeldin A-inhibited guanine nucleotide-exchange protein for adenosine diphosphate (ADP)-ribosylation factor. The isolation procedure involves homogenization of fresh bovine brain cortex, centrifugation, and three consecutive steps of column chromatography on diethylaminoethyl Sephacel and hydroxylapatite. Guanine nucleotide-exchange proteins (GEP) activation of ARF assayed by stimulation of Cholera toxin A subunit-catalyzed ADP-ribosylagmatine synthesis. GEP activity of the purified protein is assayed by its effect on ARF stimulation of CTA-catalyzed ADP-ribosylagmatine synthesis and by its effect on binding of guanosine 5'-3'-O-(thio)triphosphate (GTPyS) by native ARF1/3 purified from bovine brain cytosol. The deduced amino acid sequence of bovine BIG1 contains a central Sec7 domain (residues 697–885) and is 99% identical to human BIG1 with most differences located near the N terminus.