GTP-dependent modification of a 21-kDa substrate with NAD+ in bovine brain soluble fraction is not ADP-ribosylation of small G-protein but tailing of tRNA.
Labeling of 21-kDa material was observed when bovine brain soluble fraction was incubated with [adenylate-32P]NAD+ in the presence of GTP. The 21-kDa substrate, slightly smaller than C3 substrate in size, was labeled even without C3 exoenzyme. GTP could be replaced by nucleoside triphosphates other than ATP while ATP inhibited the GTP-induced labeling of 21-kDa substrate. After incubation of the soluble fraction with [adenylate-32P]NAD+ in the presence of GTP, [32P]ADP and [32P]ATP were detected in addition to [32P]AMP and [32P]ADP-ribose while only the last two nucleotides were observed without GTP. The 21-kDa substrate was labeled with [alpha-32P]ATP even in the absence of GTP, suggesting adenylylation rather than ADP-ribosylation. The labeled 21-kDa substrate, was extractable by phenol, disappeared with RNase treatment but not with tryptic digestion. Alkaline treatment of the phenol extract yielded an equal mixture of 3'-[32P]CMP and 2'-[32P]CMP. From these results we concluded that the 21-kDa labeling is a result of tRNA tailing with [alpha-32P]ATP generated from the [32P]AMP moiety of [adenylate-32P]NAD+. Results from reconstitution experiments using enzymes and tRNA purified from bovine brain soluble fraction, which are involved in this pathway, confirmed our conclusion.
Badruzzaman
M
M
Department of Biochemistry, Izumo, Japan.
Miyaoka
T
T
Terashima
M
M
Yamada
K
K
Ohno
T
T
Yamamori
C
C
Tsuchiya
M
M
Shimoyama
M
M
D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D012343:RNA, Transfer / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D010761:Phosphorus Radioisotopes; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins