Anion-activated 5′-nucleotidase in cell envelopes of a slightly halophilic Vibrio alginolyticus

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1. 1. Unique properties of the 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) in cell envelopes of a slightly halophilic Vibrio alginolyticus were studied. 2. 2. More than 70% of the 5′-nucleotidase activities were associated with the cell envelopes prepared by the osmotic lysis or by the lysis of penicillin spheroplasts. 3. 3. The activities in the envelopes were increased by storage in a frozen state or by pre-incubation at an alkaline pH in the presence of monovalent cations. The order of effectiveness for the pre-activation was: NH 4 + ⪢ Li + > Na + > K +  Rb +  Cs +. Such pre-activation was not detected with the envelopes prepared in the absence of Mg 2+ and Cl −. 4. 4. Both the unactivated and the activated enzymes hydrolyzed all 5′-ribonucleotides to the corresponding ribonucleosides and P 1. For the hydrolysis of these substrates, both Mg 2+ and an appropriate anion were required for maximal activity. Among monovalent anions, Cl − was the most effective, the order of effectiveness being Cl −  Br − > I − > NO 3 −. Acetate and SO 4 2− showed no effect. No essential requirement for a particular species of monovalent cation was detected. From these results, the activities in the envelopes of V. alginolyticus were identified as a Mg 2+-dependent and anion-activated 5′-nucleotidase. Furthermore, Ca 2+-dependent and anion-activated ribonucleoside 5′-monophosphatase was also found in this envelope. This preparation was unable to hydrolyze UDPG, glucose 1- and 6-phosphates, 2′- and 3′-ribonucleotides, 2′,3′-cyclic ribonucleotides, bis(p- nitrophenyl) phosphate, p- nitrophenyl phosphate or PP i. 5. 5.|The activated enzyme was inhibited by high concentrations of salts such as 1.0 M each of LiCl and NaCl. The unactivated enzyme, however, was unaffected by these salts. 6. 6.|The significance of the Cl − modifications on the enzyme activities isolated from slightly halophilic(marine) bacteria is discussed.