ADP-ribosylation of 24-26-kDa GTP-binding proteins localized in neuronal and non-neuronal cells by botulinum neurotoxin D.
Clostridium botulinum D (strain South Africa) produces ADP-ribosyltransferase which modifies eukaryotic 24-26-kDa proteins. ADP-ribosyltransferase activity was associated with a neurotoxin of 150 kDa (Dsa toxin) as confirmed by the elution profile of Dsa toxin from high performance anion-exchange column. The 24-kDa substrate of Dsa toxin-catalyzed ADP-ribosylation was detected in several tissues examined including rat brain, heart, and liver; bovine adrenal medulla; sea urchin eggs; electric organs of electric fish; and cell lines of neural (N18, N1E115, NS20Y, NG108, PC12, and C6) and non-neural (3T3) origins, suggesting its ubiquitous localization in eukaryotic cells. On the other hand, the 26-kDa substrate was detected only in membrane fractions of neural tissues and neuronal cells, suggesting its specific localization in membrane of nerve terminals. ADP-ribosylation of both the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane was potentiated by either divalent cations or guanine nucleotides, whereas adenine nucleotides did not affect the ADP-ribosylation reaction. Trypsin digestion of the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane extract produced different tryptic fragments indicative of the structural difference between the 24- and 26-kDa substrates. Both the 24- and 26-kDa substrates were less sensitive to trypsin digestion before being ADP-ribosylated by Dsa toxin than after, suggesting the conformational alterations of the 24-26-kDa proteins induced by ADP-ribosylation. These results suggest that Dsa toxin modifies two distinct low molecular mass GTP-binding proteins by ADP-ribosylation to alter their putative function(s).
Matsuoka
I
I
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Sakuma
H
H
Syuto
B
B
Moriishi
K
K
Kubo
S
S
Kurihara
K
K
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000302:isolation & purification / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D004557:Electric Organ / Q000378:metabolism; D004593:Electrophorus; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism*; D009928:Organ Specificity; D051381:Rats; D014101:Torpedo
D000227:Adenine Nucleotides; D006150:Guanine Nucleotides; D036002:ADP Ribose Transferases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins