Purification and characterization of the alpha subunit of G13.
Specific antisera were produced to peptides representing the carboxyl terminus of alpha 13, a recently identified alpha subunit of the heterotrimeric guanine nucleotide-binding 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 alpha 13 from bovine brain. Quantities of protein sufficient for biochemical analysis could be produced by concurrent expression of alpha 13 with G protein beta 2 and gamma 2 subunits using a baculovirus system. The rate of dissociation of GDP from recombinant alpha 13 (r alpha 13) is slow (0.01-0.02 min-1 at 30 degrees C), and relatively high concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) are required to observe nucleotide binding. This binding was reduced significantly in the presence of 20 mM Mg2+. Rates of hydrolysis of GTP by alpha 13 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 beta gamma subunits inhibited binding of GTP gamma S to and hydrolysis of GTP by alpha 13. By reconstitution, the purified alpha 13 did not affect the activity of several known effector enzymes. The availability of purified r alpha 13 and knowledge of its biochemical properties will allow further characterization of its interactions with receptors and effectors.
Singer
W D
WD
Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas 75235.
Miller
R T
RT
Sternweis
P C
PC
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths
D017931:DNA Primers; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins