Isolation, Characterization, and Primary Structure of a Base Non-Specific and Adenylic Acid Preferential Ribonuclease with Higher Specific Activity from Trichoderma viride

Abstract

In order to elucidate the structure-function relationship of RNases belonging to the RNase T₂ family (base non-specific and adenylic acid-preferential RNase), an RNase of this family was purified from Trichoderma viride (RNase Trv) to give three closely adjacent bands with RNase activity on slab-gel electrophoresis in a yield of 20%. The three RNases gave single band with the same mobility on slab-gel electrophoresis after endoglycosidase F digestion. The enzymatic properties including base specificity of RNase Try were very similar to those of typical T₂-family RNases such as RNase T₂ from Aspergillus oryzae and RNase M from A. saitoi. The specific activity of RNase Try towards yeast RNA was about 13-fold higher than that of RNase M. The complete primary structure of RNase Try was determined by analyses of the peptides generated by digestion of reduced and carboxymeth-ylated RNase Try with Staphylococcus aureus V8 protease, lysylendopeptidase and α-chymotrypsin. The molecular weight of the protein moiety deduced from the sequence was 25, 883. The locations of 10 half-cystine residues were almost superimposable upon those of other RNases of this family. The homologies between RNase Try and RNase T₂, RNase M, and RNase Rh (Rhizopus niveus) were 124, 132, and 92 residues, respectively. The sequences around three histidine residues, His52, His109, and His114, were highly conserved in these 4 RNases.