Subunit interactions of the Go protein.
The monoclonal antibody, MONO, recognizes an epitope on the G protein alpha o-subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTP gamma S, all beta gamma-subunits are released from the alpha o-subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTP gamma S or GTP gamma S-induced subunit dissociation. However, we have been unable to induce a similar dissociation of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent Kd of alpha o-GDP for binding beta gamma of 340-390 nM has been reported [(1989) J. Biol. Chem. 264, 20688-20696] our results would suggest that binding of MONO to the alpha o-subunit induces an increased affinity of alpha o-GDP for beta gamma. Alternatively, these results could be explained if, under the conditions used, the Kd of alpha o-GDP for beta gamma were at least two orders of magnitude lower than estimated previously.
van der Voorn
L
L
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.
Hengeveld
T M
TM
Ploegh
H L
HL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D019204:GTP-Binding Proteins / Q000276:immunology*; D006150:Guanine Nucleotides / Q000378:metabolism; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006150:Guanine Nucleotides; D019204:GTP-Binding Proteins