Cyclic 3′,5′-nucleotide phosphodiesterase of Serratia marcescens

Abstract

1. 1. A cyclic 3′,5′-nucleotide phosphodiesterase from the cells of Serratia marcescens was purified over 1000-fold. The purified preparation showed a pH maximum in the region of 7.5–8.5 in Tris-HCl buffer, with a K m of 0.52 mM for adenosine cyclic 3′,5′-phosphate. Gel filtration on a Sephadex G-100 column gave the molecular weight of 51 000. 2. 2. The esterase did not require Mg 2+ nor Mn 2 for activity, but was stimulated by Fe 2+, Ca 2+ or Ba 2+. Imidazole did not stimulate the activity. 3. 3. The cyclic 3′,5′-nucleotide phosphodiesterase of S. marcescens was inhibited competitively by theophylline. The K i value was 8.3·10 −4 s M. Proflavine, 8-hydroxyquinoline, Zn 2+, Cu 2+, Ni 2+ and 5′-deoxy-5′-(dihydroxyphosphinylmethyl)-adenosine 3′-cyclic ester were also inhibitory. The last compound was the most powerful competitive inhibitor with a K i of 4.5·10 −6 M. ADP, AMP and GTP were also slightly inhibitory. 4. 4. The enzyme was specific for nucleoside cyclic 3′,5′-monophosphates. Besides cyclic 3′,5′-monophosphates of adenosine, guanosine, inosine, thymidine and uridine the enzyme could hydrolyze cytidine cyclic 3′,5′-monophosphate at a considerable rate.