Figure 5.

The 55-kDa protein, copurified with p15, is recognized by anti-GA/1 antibodies, is ADP-ribosylated by cholera toxin, and binds GTP. (A) Affinity material was either radioiodinated and analyzed by electrophoresis and autoradiography as in Figure 3 (lane 1) or analyzed by immunoblotting with anti-GA/1 antibodies in the absence and presence of peptide GA/1 (40 μM; lanes 2 and 3, respectively). Recognition of the 55-kDa protein is indicated by a filled arrowhead. (B) Affinity material was incubated either with activated cholera toxin and [³²P]NAD, in the absence and presence of GTPγS (0.1 mM; lanes 1 and 2, respectively), or only with [³²P]NAD (lane 3), fractionated on SDS-PAGE, and gels were scanned for radioactivity with a molecular imager system. The radiolabeled 55-kDa protein is indicated by a filled arrowhead. (C) Material was either eluted with GTP (0.1 mM in the presence of 0.5 mM MgCl₂) from affinity chromatography loaded with membrane preparations previously not incubated or incubated with GTPγS (0.1 mM; lanes 1 and 2, respectively) or with ATP (0.1 mM in the presence of 0.5 mM MgCl₂) from affinity chromatography loaded with membrane preparations not incubated with GTPγS (lane 3), and then analyzed by immunoblotting with anti-GA/1 antibodies. The 55-kDa protein detected in lane 1, and not detected in the two other lanes, is indicated by a filled arrowhead. (D) Material was eluted with [α-³²P]GTP (16 nM, 3000 Ci/mmol) from affinity chromatography, exposed to UV irradiation, fractionated on SDS-PAGE, and blotted onto polyvinylidene difluoride filters. Blotted filters were then exposed to autoradiography (lane 1) or incubated with anti-GA/1 antibodies (lane 2). Complexes between [α-³²P]GTP and the 55-kDa protein are indicated by filled arrowheads; two other minor species of radiolabeled complexes, not recognized by anti-GA/1 antibodies, are indicated by open arrowheads.