Purification and properties of an (ADP-ribose)n glycohydrolase from guinea pig liver nuclei.
An (ADP-ribose)n glycohydrolase has been purified more than 3,000-fold from guinea pig liver nuclei with an 18% yield. The glycohydrolase activity present in the nuclei was solubilized only by sonication at high ionic strength and purified by sequential chromatographic steps on phosphocellulose, DEAE-cellulose, Blue Sepharose, and single-stranded DNA cellulose. The purified protein exhibited one predominant protein band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 75,500. On Sephadex G-100 gel filtration, single coincident peaks of (ADP-ribose)n glycohydrolase activity and protein with a molecular weight value of 72,000 were observed. The Km value for (ADP-ribose)n and the maximal velocity of the highly purified glycohydrolase were 2.3 microM and 36 mumol of ADP-ribose released from (ADP-ribose)n . min-1 . mg protein-1, respectively. Hydrolysis of (ADP-ribose)n by the enzyme was exoglycosidic in nature. The optimum pH for the enzyme activity was apparent at 6.8-7.0. Sulfhydryl compounds and monovalent cations were required for the maximal activity. The enzyme was sensitive to Ca2+ but not to Mg2+. The enzyme activity was inhibited by ADP-ribose, cyclic AMP (adenosine 3':5'-monophosphate) and diadenosine 5',5'''-p1,p4-tetraphosphate. Denatured DNA and histones were inhibitory, but native DNA and its histone complex were not inhibitory. Our data indicate that the glycohydrolase is present only as a minor protein in nuclei, being present in perhaps about 50,000 molecules/nucleus.
D000818:Animals; D002412:Cations / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004247:DNA / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006168:Guinea Pigs; D006657:Histones / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000166:cytology*; D013010:Sonication; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002412:Cations; D006657:Histones; D013438:Sulfhydryl Compounds; D004247:DNA; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase