Peptide mapping studies of the pertussis toxin substrate in human neutrophils, platelets and erythrocytes.
Protease digestion of the ADP-ribosylated pertussis toxin substrate (PTS) protein was carried out after solubilization with SDS (Cleveland gels) and in the intact membrane. Cleveland gel analysis showed substantial similarities in the maps for the PTS component in neutrophils, platelets and erythrocytes and also in the S49 AC-lymphoma cell line. In the intact membrane ADP-ribosylation followed by digestion showed limited access of proteases to the PTS component. Of eight proteases tested, only papain and Staphylococcus aureus gave substantial digestion. This pattern was observed in the human platelet, erythrocyte and neutrophil plasma membranes. When the sequence was reversed and ADP-ribosylation was carried out after protease digestion, a very different pattern was observed with much greater susceptibility to digestion being noted with several proteases. By contrast, analysis of the murine AC-membrane showed some minor variations in the digest patterns. In addition, under all three conditions tested, maps of the cholera toxin substrate for the human platelet showed remarkable similarities to those obtained with the pertussis toxin substrate. Our results indicate that the protease sensitive sites of the alpha subunit of PTS and protection from proteolysis after ADP-ribosylation are properties which are shared by the PTS components of human platelets, erythrocytes and neutrophils.
Scott
S J
SJ
Kaiser Regional Research Laboratory, Los Angeles, CA 90027.
Olson
C V
CV
Lad
P M
PM
D000246:Adenosine Diphosphate Ribose / Q000494:pharmacology; D001792:Blood Platelets / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis*; D009504:Neutrophils / Q000032:analysis*; D010447:Peptide Hydrolases / Q000494:pharmacology; D010449:Peptide Mapping; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D008565:Membrane Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D010447:Peptide Hydrolases