Identification of a new GTP-binding protein. A M(r) = 43,000 substrate for pertussis toxin

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg²⁺ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 μM. Addition of a 10-fold molar excess of purified βγ subunits (M(r) = 35,000 β; and M(r) = 7,000 γ) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl₂ resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the α subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S.M., Kahn, R.A., Manning, D.R., and Gilman, A.G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH₄C₁, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.