Cytosol promotes the guanine nucleotide-induced release of the alpha subunit of Gi2 from the membrane of mouse mastocytoma P-815 cells.
Translocation of the alpha subunit of Gi2 from the membrane to the cytosol was studied in mouse mastocytoma P-815 cells. To monitor Gi2 alpha the membrane (300,000 x g pellet) was [32P]ADP-ribosylated with pertussis toxin. Incubation of the [32P]ADP-ribosylated membrane with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a small release (10%) of [32P]ADP-ribosylated Gi2 alpha from the membrane. Whereas cytosol (300,000 x g supernatant) alone had no ability to release the [32P]ADP-ribosylated Gi2 alpha from the membrane, it markedly augmented the release induced by GTP gamma S, about 50% of the total [32P]ADP-ribosylated Gi2 alpha being released by 30 min. The GTP gamma S-induced release and its enhancement by the cytosol were specific for GTP and GTP gamma S. When the cytosol was boiled this promoting activity was lost. The [32P]ADP-ribosylated Gi2 alpha released by the cytosol plus GTP gamma S from the membrane was eluted as a single peak corresponding to a molecular weight of about 100,000 from an Ultrogel AcA 44 column. In contrast, the [32P]ADP-ribosylated Gi2 alpha released by GTP gamma S alone was eluted at the position of Mr = 40,000, but it was eluted at the position of Mr = about 100,000 when it was incubated with the cytosol. Furthermore, Gi2 alpha purified from bovine lung also behaved in a similar way on gel filtration. The addition of thrombin, a stimulant of histamine secretion from mast cells, to mastocytoma cells drastically induced the translocation of Gi2 alpha from the membrane to the cytosol in a pertussis toxin-sensitive manner. These results taken together demonstrate that the cytosol contains some factor(s) that promotes the release of GTP-activated Gi2 alpha from the membrane and that the released Gi2 alpha exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.
Takahashi
S
S
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Negishi
M
M
Ichikawa
A
A
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002850:Chromatography, Gel; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D012515:Mast-Cell Sarcoma / Q000378:metabolism*; D051379:Mice; D037342:Pertussis Toxin; D013379:Substrate Specificity; D013917:Thrombin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006150:Guanine Nucleotides; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D013917:Thrombin