ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A1.
We have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S.GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S.GTP gamma S), forms over a period of 10-15 min at 37 degrees C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S.GTP gamma S forms in membranes that are exposed to CF alone and then to GTP gamma S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S.GTP gamma S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45 degrees C is decreased by GTP gamma S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. We suggest that active S interacts directly with the enzymic A1 fragment of cholera toxin and not with any toxin substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Gill
D M
DM
Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, Massachusetts.
Coburn
J
J
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002772:Cholera Toxin / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D013873:Thionucleotides; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D002772:Cholera Toxin