Purification, characterisation, and molecular cloning of a chicken erythroblast mono(ADP-ribosyl)transferase.
We have purified an arginine-specific mono(ADP-ribosyl)transferase from chicken erythrocytes. The purified transferase was free from poly (ADP-ribose) polymerase activity. The molecular weight of the purified enzyme was estimated to be 27.5 kDa by gel filtration through Sephadex G-75 in a non-denaturing solvent. Activity gel experiments indicate that the active enzyme has an apparent molecular weight in SDS gels of about 28 kDa. The optimum pH of the reaction is about 8.0. The K(m) value for NAD+ of the purified enzyme is about 130 microM. Small molecular weight inhibitors of poly (ADP-ribose) polymerase have no significant effect on the mono ADP-ribosyl transferase enzyme activity. A number of inhibitors of the arginine-specific mono(ADP-ribosyl)transferase activity have been identified. Among the more effective inhibitors are 1,4 naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 4-amino-1-naphthol and 1,2-naphthoquinone. We have also cloned a mono(ADP-ribosyl)transferase from chicken erythroblasts. This gene has been expressed in E. coli and ADP-ribosylation activity has been demonstrated using histones as substrate. The activity is shown to be arginine-specific by the use of poly-L-arginine as substrate. Use of a specific inhibitor has shown that this enzyme is indeed a mono(ADP-ribosyl)transferase and not a NAD glycohydrolase activity. The sequence of this gene is very similar to several other mono(ADP-ribosyl)transferase genes. There are thus at least three different chicken mono(ADP-ribosyl)transferase genes in the blood system alone; this suggests that there is a quite large family of mono(ADP-ribosyl)transferase genes in animals. We have also isolated the promoter region of this chicken gene and are able to identify several standard motifs in this promoter.
Davis
T
T
Cell and Molecular Biology Laboratory, School of Biological Sciences, University of Sussex, Brighton, England.
Sabir
J S
JS
Tavassoli
M
M
Shall
S
S
D036002:ADP Ribose Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004900:Erythroblasts / Q000201:enzymology*; D006657:Histones / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D006657:Histones; D001120:Arginine; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases