Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line

The pertussis toxin substrate from RAW264 macrophage cell membranes was characterized by two-dimensional gel electrophoresis and by immunoblots using antibodies directed against different guanine nucleotide binding proteins. RAW264 membranes were found to contain one major pertussis toxin substrate, which was recognized by both antibodies AS/6 and LE/3. The AS/6 antibody was made against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of transducin, and the LE/3 antibody was made against the peptide corresponding to amino acids 160-169 of a guanine nucleotide binding protein (Gi-2-alpha) cloned from a mouse macrophage cell line. The RAW264 pertussis toxin substrate was not recognized by either antibody CW/6 or antibody RV/3, which recognize the 41-kilodalton alpha-subunit of brain Gi (Gi-1-alpha) and Go-alpha, respectively. Pertussis toxin substrates from bovine brain were resolved into four major alpha-subunits by two-dimensional gel electrophoresis, and the LE/3 antibody recognized only one of the four proteins. The brain LE/3 reactive protein also reacted with the AS/6 antibody, migrated with a 40K molecular weight, and had an isoelectric point slightly more basis than the RAW264 pertussis toxin substrate. Therefore, the major pertussis toxin substrate in RAW264 cells appears to be Gi-2, and bovine brain contains a relatively minor amount of a closely related guanine nucleotide binding protein.