Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)

Ribonucleases with antitumor activity are mainly found in the oocytes and e mbryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolate d from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNas e 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by c olumn chromatographs and detected by zymogram assay and western blotting. C haracterization of these purified ribonucleases revealed that they were hig hly conserved in amino acid sequence and had a pyroglutamate residue at the ir N-termini, but possessed different specific activities, base specificiti es and optimal pH values for their activities. These ribonucleases were cyt otoxic to cervical carcinoma Hela cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to b e involved in the cytotoxicity of the frog ribonucleases to tumor cells. Be cause the coding regions lack introns, the ribonuclease genes were cloned b y PCR using genomic DNA as template. Their DNA sequences and amino acid seq uences are homologous to those of mammalian ribonuclease superfamily, simil ar to 50 and similar to 25%, respectively.