Specific antisera were produced to peptides representing the carboxyl terminus of α13, a recently identified α subunit of the heterotrimeric guanine nucleotidebinding proteins (G proteins). Immunodetection with the antisera indicated that the 43-kDa protein is expressed ubiquitously at low levels (0.005-0.05% of membrane protein) in tissues and cultured cells. A combination of conventional and immunoaffinity Chromatographic techniques was used to purify small quantities of α13 from bovine brain. Quantities of protein sufficient for biochemical analysis could be produced by concurrent expression of α13 with G protein β2 and γ2 subunits using a baculovirus system. The rate of dissociation of GDP from recombinant α13 (rα13) is slow (0.01-0.02 min-1 at 30 °C), and relatively high concentrations of guanosine 5′-3-O-(thio)triphosphate (GTPγS) are required to observe nucleotide binding. This binding was reduced significantly in the presence of 20 mM Mg2+. Rates of hydrolysis of GTP by α13 were limited by nucleotide exchange; attempts to measure the intrinsic rate of hydrolysis indicate that it is greater than 0.2 min-1. Stoichiometric concentrations of βγ subunits inhibited binding of GTPγS to and hydrolysis of GTP by α13. By reconstitution, the purified α13 did not affect the activity of several known effector enzymes. The availability of purified rα13 and knowledge of its biochemical properties will allow further characterization of its interactions with receptors and effectors.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Aug 5 1994|
ASJC Scopus subject areas