EXTRACELLULAR RIBONUCLEASE FROM BACILLUS POLYMYXA : ISOLATION, STRUCTURE, AND FUNCTIONING

A preparative-scale procedure was developed to isolate three individual forms (BpoI, BpoII, and BpoIII) of the extracellular cyclizing ribonuclease from Bacillus polymyxa, providing respectively 3%, 21%, and 2% yield with 5275-, 7275- and 6950-fold purification, Relative molecular masses, amino acid composition, N- and C-terminal amino acid sequences were determined for these three forms. Two forms of the enzyme, BpoI and BpoIII containing additional segments of 12 and 6 residues at the N end were shown to be pro-forms of the mature enzyme, BpoII. The N-terminal amino acid sequence of B. polymyxa RNase was different from those found in Bacillus RNases studied earlier; however common features indicated the same protein family. The ability of B. polymyxa RNase to hydrolyze dinucleoside diphosphates and polynucleotides was maximal for the substates containing guanosine or its derivatives. The enzyme was inhibited by specific complex formation (dissociation constant 2.7 . 10(-12) M) with barstar, a natural inhibitor of B. amyloliquefaciens RNase.