Two lectins with RNase activity obtained from eggs of Rana catesbeiana and R. japonica and RNase obtained from R. catesbeiana liver show 65-83% protein homology. The base specificity of these frog proteins was studied with 8 dinucleoside phosphates as substrates and 8 nucleotides as inhibitors. The base specificities of the B1 and B2 sites of these proteins are U greater than C and G greater than U greater than A, C, respectively. The three frog proteins are more resistant than RNase A to heat treatment, guanidine-HCl and pH-induced denaturation; i.e., they retain their native conformation up to at least 70 degrees C at pH 7.5. Differences in stability and base specificity among RNase A and the three frog proteins are discussed in relation to the primary structures. Although the two lectins agglutinate tumor cells (e.g., Ehrlich, S-180 and AH109A ascites carcinoma cells), the liver RNase has no such activity. Agglutination of AH109A cells by the two lectins is inhibited by nucleotides. Our results indicate that the agglutination sites are not identical with, but are related to, the active sites of the three frog proteins.
A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.
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