The specific activity of rat poly(adenosine diphosphate ribose) glycohydrolase was higher in the testis than in the liver, brain, spleen or kidney. The enzyme was found primarily in the soluble fraction of the testis. When the soluble enzyme was chromatographed on phosphocellulose, the activity eluted in two peaks, at 0.22 and 0.34 m KCl, respectively, referred to in the present study as enzyme A and B. Enzyme A has an optimal pH of 7.25 and was stimulated by 150 mm KCl. The optimal pH of enyzme B was 6.5, but it was not stimulated by KCl. For maximal activity both enzymes required 10 mm 2-mercaptoethanol, and they were strongly inhibited by 100 μm p-chloromercuribenzoate. The Km values of enzyme A and B for poly(adenosine diphosphate ribose) were 1.52 and 0.70 μm, respectively. Ribose 5′-phosphate, guanosine 3′,5′-monophosphate, adenosine 3′,5′-monophosphate and adenosine diphosphate ribose inhibited both enzymes. The two latter nucleotides behave as noncompetitive inhibitors. Denatured DNA and the homopolypurines poly(G), poly(I) and poly(A) were very potent inhibitors of both glycohydrolases. The mode of hydrolysis of poly(adenosine diphosphate ribose) by glycohydrolases A and B was exoglycosidic, yielding adenosine diphosphate ribose as the final product.
ASJC Scopus subject areas
- Molecular Biology