Biochemical discrimination of the predominant pertussis toxin substrate of rabbit neutrophils from brain Gi and Go: isoelectric focusing improves resolution of pertussis toxin substrates
Kanaho, Y.; Stadel, J.M.; Huang, C.K.; Becker, E.L.
Biochemistry International 16(5): 879-886
1988
ISSN/ISBN: 0158-5231 PMID: 3138999 Document Number: 321299
In the present study, the predominant pertussis toxin substrate in rabbit neutrophilis, Gn, was biochemically compared to G1 and Go purified from brain, after being [32P]ADP-ribosylated by activated pertussis toxin and [32P]NAD. On SDS-polyacrylamide gels, a poorly resolved doublet from neutrophil membranes was observed; the upper band, correponding to .simeq.25% labeling, comigrated with G1-.alpha. and the predominant lower band, Gn, migrated intermediately between G1-.alpha. and Go-.alpha. Peptide maps generated by limited-digestion of the labeled Gn, G1 and Go with S. aureus V8 protease were slightly, but definitively and reproducibly different. Isoelectric focusing clearly distinguished Gn from the other two pertussis toxin substrates. The pI value of Gn, 5.60, was distinctly different from those of G1, 5.75 and 5.80. Although the pI values for Go and Gn were similar (5.60), the patterns of the two proteins were qualititatively different, with Go being resolved into an equal doublet (pI=5.55 and 5.60) while Gn appeared predominantly as a single band. Thus, Gn is biochemically distinguishable from G1 and Go of brain and these structural differences are most clearly evident following isoelectric focusing.