MOLECULAR CHARACTERIZATION OF THE INVITRO ACTIVATION OF PERTUSSIS TOXIN BY ATP

Pertussis toxin (PT)-catalyzed ADP-ribosylation of transducin (G(t)) i s stimulated by ATP. In the absence of ATP, PT exhibited an approximat ely 20-fold lower linear velocity than the recombinant S1 subunit (rS1 ) in catalyzing the ADP-ribosylation of G(t). In the presence of 0.1 m M ATP, the linear velocities of rS1 and PT were essentially identical. ATP increased the k(cat) of PT-catalyzed ADP-ribosylation of G(t) wit hout altering the K(mapp) for either G(t) or NAD. Further, in the pres ence of ATP, PT exhibited similar kinetic constants under conditions o f variable G(t) and variable NAD as rS1 in catalyzing the ADP-ribosyla tion of G(t). The S1 subunit of PT was cleaved by chymotrypsin to a si ngle immunoreactive peptide in the absence of ATP, while three immunor eactive peptides were generated in the presence of ATP. The S1 subunit of PT was not cleaved by trypsin in the absence of ATP, at the concen trations of trypsin used, while two immunoreactive peptides were produ ced in the presence of ATP. The immunoreactive peptides produced eithe r by chymotrypsin or trypsin cleavage of the S1 subunit of PT in the p resence of ATP were indistinguishable from those produced by cleavage of rS1 with the same protease. Chymotryptic and tryptic cleavage of rS 1 was not altered by ATP. When PT was incubated with ATP prior to Bio- Gel P-100 gel filtration, approximately 8% of the S1 subunit dissociat ed from the B oligomer, as determined by ADP-ribosyltransferase assays of the column eluant. This increased to 20% when ATP was included in the column buffer. The presence of dithiothreitol and NAD in addition to ATP did not affect the amount of dissociated S1 subunit. Our data f urther indicated that activation of PT by ATP was a reversible process . Together, these data showed that ATP quantitatively converted the S1 subunit of PT to a form which was kinetically and conformationally id entical with rS1, while only a fraction of the S1 subunit was dissocia ted from the B oligomer. These results indicate that both S1 subunit w hich is bound to the B oligomer as well as dissociated S1 subunit are capable of catalyzing the ADP-ribosylation of G(t).