Characterization of the putative alpha subunit of a heterotrimeric G protein in rice.
A recombinant protein with a cDNA that encodes the putative alpha subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice alpha) with an apparent molecular mass of 45 kDa was bound with guanosine 5'-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its kcat was 0.44. rGrice alpha was ADP-ribosylated by activated cholera toxin. Monoclonal antibodies raised against rGrice alpha reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice alpha. A 45 kDa polypeptide in the plasma membrane, as well as rGrice alpha, was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the alpha subunits of heterotrimeric G proteins in animals, suggesting that the putative alpha subunit is truly the alpha subunit itself.
Iwasaki
Y
Y
Department of Bioscience, Faculty of Biotechnology, Fukui Prefectual University, Japan.
Kato
T
T
Kaidoh
T
T
Ishikawa
A
A
Asahi
T
T
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000918:Antibody Specificity; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology; D002772:Cholera Toxin / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020690:GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D012275:Oryza / Q000737:chemistry / Q000235:genetics; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C085379:rho GTPase-activating protein; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins