Purification and Properties of a Glycohydrolase from Calf Thymus Splitting Ribose-Ribose Linkages of Poly(Adenosine Diphosphate Ribose)

Abstract

Abstract A new enzyme splitting the ribose-ribose bonds of poly-(adenosine diphosphate ribose) was purified 200-fold from calf thymus. The molecular weight of the enzyme was estimated to be 48,000 by gel filtration and 53,000 by sucrose density gradient centrifugation. Its optimum pH was around 7.5. For full activity 10 mm 2-mercaptoethanol or dithiothreitol was required. Activity was inhibited considerably by 10 µm p-chloromercuriphenylsulfonate or HgCl2. The Km value for poly(adenosine diphosphate ribose) was 0.58 µm. Adenosine cyclic 3' : 5'-monophosphate and adenosine diphosphate ribose inhibited the reaction, causing 50% inhibition at concentrations of 0.3 mm and 1.1 mm, respectively. Calf thymus histone f2a, f2b, and f3, protamine, and poly-l-lysine inhibited the activity. The inhibition by histone f2a was reversed by DNA. The enzyme hydrolyzed poly(adenosine diphosphate ribose) in a fashion of an exoglycosidase. The end product of hydrolysis is adenosine diphosphate ribose. This enzyme may function in regulation of the chain length of poly(adenosine diphosphate ribose), which was previously claimed to play a role in DNA synthesis or in the structure of chromatin.