Activator protein supporting the botulinum ADP-ribosyltransferase reaction.
The ADP-ribosyl moiety of NAD was transferred to proteins with Mr values of 22,000 and 25,000 when bovine brain cytosol was incubated with a botulinum ADP-ribosyltransferase C3 (BT-C3) which was purified from the culture medium of a type C strain of Clostridium botulinum. Any protein fraction eluted from a chromatographic column to which the cytosol had been applied, however, was not significantly ADP-ribosylated by BT-C3, unless the reaction mixture was further supplemented with a small amount of the cytosol. Thus, substrate protein(s) could be partially purified based on their ability to be ADP-ribosylated by BT-C3 in the presence of the cytoplasmic activator(s). The rate of ADP-ribosylation of the substrates was extremely low by itself but was increased enormously and progressively when increasing amounts of cytosol were added, affording a reliable means for assay of the activator contained therein. The activator was separated from the substrate proteins and partially purified from the cytosol by sequential chromatography steps with an anion exchanger and a gel filtration column. The activity of the partially purified activator was heat-labile and protease-sensitive, suggesting that the activator was a protein or had a protein component necessary for activity. The action of the activator protein(s) was specific for BT-C3-catalyzed ADP-ribosylation; cholera toxin-catalyzed ADP-ribosylation of GTP-binding protein (Gs) was not supported by this activator. Thus, this is the first report to show that botulinum ADP-ribosyltransferase-catalyzed reaction can proceed significantly only in the presence of other protein factor(s), just as has been observed with an ADP-ribosylation factor required for cholera toxin-induced similar reaction.
Ohtsuka
T
T
Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Yokohama, Japan.
Nagata
K
K
Iiri
T
T
Nozawa
Y
Y
Ueno
K
K
Ui
M
M
Katada
T
T
D036002:ADP Ribose Transferases / Q000378:metabolism*; D020727:ADP-Ribosylation Factors; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology*; D001921:Brain / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000502:physiology*; D013379:Substrate Specificity
D008565:Membrane Proteins; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors