Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose).
Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.
D000818:Animals; D002417:Cattle; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D046911:Macromolecular Substances; D009702:Nucleoside Diphosphate Sugars; D011064:Poly Adenosine Diphosphate Ribose; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase