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Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog)
1Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei 100, Taiwan. ydliao@ibms.sinica.edu.tw
Abstract:
Ribonucleases with antitumor activity are mainly found in the oocytes and embryos 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 isolated 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-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic 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 be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.
Insights
Bullfrog ribonucleases isolated from oocytes and liver exhibit antitumor activity against cancer cells. Their cytotoxicities are not directly linked to enzymatic activity, suggesting other factors contribute to their effectiveness.
Area of Science:
- Biochemistry
- Molecular Biology
- Developmental Biology
Background:
- Ribonucleases with antitumor properties are primarily found in frog oocytes and embryos.
- The specific roles of these ribonucleases in frog development remain largely unknown.
- Most frog ribonuclease genes have not been cloned or characterized.
Purpose of the Study:
- To isolate and characterize ribonucleases from Rana catesbeiana (bullfrog).
- To investigate the antitumor activity and cytotoxic mechanisms of these frog ribonucleases.
- To clone and analyze the genetic sequences of these ribonuclease genes.
Main Methods:
- Purification of ribonucleases using column chromatography.
- Detection via zymogram assay and western blotting.
- Cloning of ribonuclease genes using PCR with genomic DNA.
Main Results:
- Six oocyte ribonucleases (RC-RNase to RC-RNase 6) and one liver ribonuclease (RC-RNase L1) were purified.
- These ribonucleases showed conserved amino acid sequences, N-terminal pyroglutamate, and varied specific activities and optimal pH.
- All purified ribonucleases were cytotoxic to HeLa cells, but cytotoxicity did not correlate with enzymatic specific activity.
- Ribonuclease genes, lacking introns, showed homology to mammalian ribonuclease superfamily sequences.
Conclusions:
- Frog ribonucleases possess antitumor activity, with cytotoxicity potentially involving non-catalytic residues.
- The characterized bullfrog ribonucleases are homologous to the mammalian ribonuclease superfamily.
- Further research is needed to elucidate the precise roles of these ribonucleases in frog development and their therapeutic potential.