Structural studies of cytotoxic ribonucleases from Rana catesbiana.

Lih-Woan Chen1, Chun-Hua Hsu1,2, Ning-Yuan Su1, Iren Wang1,2, Tung-Liang Lee1,3, You-Di Liao1, Chinpan Chen1.1Insititute of Biomedical Sciences, Academia Sinica, 2Institute of Biochemical Sciences, National Taiwan University, and 3Institutes of Life Sciences and Biochemistry, National Defense Medical Center, Taipei, Taiwan, R.O.C.

Recently, seven ribonucleases from Rana catesbiana (bullfrog) oocytes and liver tissues have been isolated and purified via ion exchange chromatography. Previous studies showed that the occurrence of several homologous ribonucleases in different tissues of the same animal suggests that the existence of a family of homologous genes expresses in tissue-specific fashion. Most mammalian ribonucleases are not cytotoxic to tumor cells, but most oocyte ribonucleases from frog Rana spp. possess cytotoxicity toward tumor cells, including these seven ribonucleases from Rana catesbiana. At the present time, we’ve known that these seven ribonucleases possess different ribonucleolytic activity, base specificity, and cytotoxicity. In order to know the structure-function relationships on these ribonucleases obtained from Rana catesbiana, we have used molecular biological, biochemical, and biophysical methods to carry out the structural characteristics on these ribonucleases. We have expressed the unlaebled and 15N, 13C-labeled recombinant proteins for some of the frog ribonucleases. Circular dichroism (CD) spectrum is employed to investigate the secondary structure, thermostability, and conformational change of the ribonucleases at different pH values. In addition, the stopped-flow CD and fluorescence are used in approaching on the kinetics of structural transitions of proteins. NMR techniques are applied to determine the protein structures, folding and dynamics. The preliminary structural study on these frog ribonucleases is discussed.