Purification of poly(ADP-ribose) glycohydrolase and detection of its isoforms by a zymogram following one- or two-dimensional electrophoresis.
Poly(ADP-ribosyl)ation metabolism, a post-translational modification, involves two nuclear enzymes. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are responsible for the anabolism and catabolism of poly(ADP-ribose) polymer, respectively. PARG, despite being less abundant than PARP, is a crucial determinant of polymer metabolism which is known to be implicated in DNA repair and other cellular processes. Here, we describe modifications to improve the purification of PARG from calf thymus, in terms of both quantity and quality, which would allow biochemical and immunological studies. We also developed a zymogram to identify functional polypeptides exhibiting PARG activity. Purified and crude enzyme preparations from calf thymus were electrophoresed in two-dimensional gels. Samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing the polymer substrate in the form of automodified PARP after a nonequilibrium pH gradient electrophoresis. After renaturation of PARG in the gel, four isoforms of activity were clearly detected in the purified enzyme preparation. Even in the crude extract of the tissue, we could observe the major isoform of PARG. This technique will permit a better understanding of poly(ADP-ribose) catabolism and better characterization of PARG isoforms.
Brochu
G
G
Molecular Endocrinology Laboratory, CHUL Research Center, Sainte-Foy, Québec, Canada.
Shah
G M
GM
Poirier
G G
GG
D000818:Animals; D002417:Cattle; D003101:Collodion; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D012680:Sensitivity and Specificity; D013950:Thymus Gland / Q000201:enzymology
D007527:Isoenzymes; D003101:Collodion; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase