Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells.
We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.
Takahashi
S
S
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Hashida
K
K
Yatsunami
K
K
Fukui
T
T
Negishi
M
M
Katada
T
T
Ui
M
M
Kanaho
Y
Y
Asano
T
T
Ichikawa
A
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry; D002850:Chromatography, Gel; D003600:Cytosol / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008407:Mast Cells / Q000737:chemistry*; D012515:Mast-Cell Sarcoma; D051379:Mice; D037342:Pertussis Toxin; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D000244:Adenosine Diphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins