Isolation and cloning of rat poly(ADP-ribose) glycohydrolase: presence of a potential nuclear export signal conserved in mammalian orthologs.
Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme of poly(ADP-ribose) degradation. To understand its structure-and-function relationship, we purified Parg from rat testis 9,740-fold using an improved affinity column; the purified product was a 60 kDa protein. Based on the determined sequences of three peptide fragments, degenerated primers were synthesized and a Parg cDNA comprising 3,974 nucleotides, encoding a 109 kDa protein, was isolated. The 60 kDa Parg purified from rat testes corresponded to the C-terminal half of the 109 kDa deduced peptide. When recombinant rat Parg was expressed as a glutathione S-transferase fusion protein in Escherichia coli, Parg activity was observed for the full-length and C-terminal half proteins but not in for the N-terminal half protein. Taken together, these data indicate that the catalytic domain of Parg is located in the C-terminal half. Further, we newly identified the presence of a potential nuclear export signal in the N-terminal half in addition to the previously reported nuclear localization signals in rat and other mammalian Pargs. Northern blot analysis showed the ubiquitous expression of a single 4.0 kb Parg mRNA in various rat tissues. The findings suggest that the 60 kDa Parg is produced by post-transcriptional processing.
Shimokawa
T
T
Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, 104-0045, Japan.
Masutani
M
M
Nagasawa
S
S
Nozaki
T
T
Ikota
N
N
Aoki
Y
Y
Nakagama
H
H
Sugimura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013737:Testis / Q000201:enzymology; D014018:Tissue Distribution
D017931:DNA Primers; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase