Low-molecular-weight GTP-binding proteins serving as ADP-ribosylation substrate for ADP-ribosyltransferase from Clostridium botulinum and their relation to phosphoinositides metabolism in thymocytes.
ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P. et al. (1987) J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.
Wang
P
P
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
Nishihata
J
J
Makishima
F
F
Moriishi
K
K
Syuto
B
B
Toyoshima
S
S
Osawa
T
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D003014:Clostridium botulinum / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism*
D010716:Phosphatidylinositols; D000244:Adenosine Diphosphate; D011065:Poly(ADP-ribose) Polymerases; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase