Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit.
Trypsin digestion of pertussis toxin (PT) preferentially cleaved the S1 subunit at Arg-218 without detectable degradation of the B oligomer. The fragment produced, termed the tryptic S1 fragment, appears to remain associated with the B oligomer. Chymotrypsin digestion of PT also preferentially cleaved the S1 subunit without detectable degradation of the B oligomer. The chymotryptic S1 fragment possessed a slightly lower apparent molecular weight than the tryptic S1 fragment and was more accessible to the respective protease. Trypsin- and chymotrypsin-treated PT and PT required the presence of dithiothreitol and ATP for optimal enzymatic activity. Trypsin-treated PT showed approximately a 2-4-fold higher level of expression of ADP-ribosyltransferase and NAD-glycohydrolase activities than PT. Chymotrypsin-treated PT also exhibited approximately a 2-fold greater level of ADP-ribosyltransferase activity than PT. The observed increase in activity of protease-treated PT was due primarily to a shorter time for activation in PT mediated ADP-ribosylation of transducin. In addition, trypsin-digested PT possessed the same cytotoxic potential for Chinese hamster ovary cell clustering as PT. One possible role for the generation of a proteolytic fragment of the S1 subunit of PT would be to produce a catalytic fragment with increased efficiency for ADP-ribosylation of G proteins in vivo.
Krueger
K M
KM
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Mende-Mueller
L M
LM
Barbieri
J T
JT
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002449:Cell Aggregation; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D006224:Cricetinae; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009244:NAD+ Nucleosidase / Q000378:metabolism; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015293:Transducin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D010566:Virulence Factors, Bordetella; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase; D002918:Chymotrypsin; D014357:Trypsin; D015293:Transducin