ATP-sensitive ribonuclease of Bacillus cereus.

Intracellular predominant ribonuclease (EC 3.1.27.2) in the stationary phase cells of Bacillus cereus IF0 3131 was purified about 3,400-fold to a homogeneous state. The ribonuclease was identified to be of the nonspecific cyclizing type, devoid of cyclic phosphodiesterase activity, as the final reaction products proved to be nucleoside 2',3'-cyclic monophosphates. Molecular weight was estimated at 28,000 by gel filtration, sodium dodecy1 sulfate-gel electrophoresis, and amino acid analysis. The antiserum, against the purified ribonuclease, cross-reacted with those of the B. cereus group which had optimum pH at 6.8, but did not react with similar enzymes of the Bacillus subtilis group which possessed optimum pH at 5.7. A feature discovered in the enzyme was the high and selective sensitivity to ATP. The ribonuclease was inhibited to more than 99% in the presence of 1 PM ATP when assayed by spectrophotometric measurement of an acid-soluble fraction using yeast RNA as substrate. The mode of inhibition by ATP was competitive, and the Ki was 10 rn at pH 6.8. An endonucleolytic activity, however, could be detected even in the presence of 1 m~ ATP by polyacrylamide gel electrophoresis using ribosomal RNA of B. cereua as substrate. It was revealed that ATP had only reduced the reaction velocity to about '/eo in the presence of 20 PM ATP with no accompanying change of substrate specificity. These results indicate that the ribonuclease cannot be completely inhibited by the physiological concentration of ATP in the stationary phase cells of B. cereus and may have participated in the endonucleolytic degradation of ribosomal RNA.