Characterization of cytosolic pertussis toxin-sensitive GTP-binding protein in mastocytoma P-815 cells

Abstract

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 hemogenate was detected in the supernatant after centrifugation at 100 000 × g for 90 min. [ 32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous βγ complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ulrogel AcA 44 column, but the βγ complex was not detected in the cytosol by using the anti-βγ complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [ 35S]GTPγS binding was not inhibited by GDP and [ 32P]ADP-ribosylation was not affected by GTPγS treatment. Only after the cytosolic PT substrate had been mixed with exogenous βγ compleex, did it copurify with exogenous γψ complex several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2α by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2α without βγ complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells.