Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.
The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.
Watkins
P A
PA
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
Kanaho
Y
Y
Moss
J
J
D036002:ADP Ribose Transferases; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D009243:NAD / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010430:Pentosyltransferases / Q000097:blood / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D015293:Transducin; D014166:Transferases / Q000494:pharmacology; D014422:Turkeys
D008565:Membrane Proteins; D009711:Nucleotides; D009243:NAD; D000244:Adenosine Diphosphate; D014166:Transferases; D036002:ADP Ribose Transferases; D010430:Pentosyltransferases; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D015293:Transducin