Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus.
Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006657:Histones / Q000494:pharmacology; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D011073:Polyamines / Q000494:pharmacology; D013552:Swine; D013950:Thymus Gland / Q000201:enzymology*
D006657:Histones; D009711:Nucleotides; D011073:Polyamines; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase