LARGE-SCALE PREPARATION OF A RIBONUCLEASE FROM RANA-CATESBEIANA (BULLFROG) OOCYTES AND CHARACTERIZATION OF ITS SPECIFIC CYTOTOXIC ACTIVITY AGAINST TUMOR-CELLS

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine seque nce-specific ribonuclease found only in R. catesbeiana (bullfrog) oocy tes, but not in other organs. The protein is localized in the yolk gra nules of oocytes but not in other organelles, as de tected by immunohi stochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk gra nules, extraction of RC-RNase with 0.09 M NaCl, selective removal of i mpurities by Hepes buffer, and chromatographies on phosphocellulose an d carboxymethyl cellulose columns. Three milligrams of RC-RNase was pu rified from a l-g pellet of yolk granules prepared from 2 g of ovary t issue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tiss ue. The properties of RC-RNase isolated from yolk granules tested so f ar are identical to those of RC-RNase isolated from the cytosolic frac tion and similar to those of a sialic acid-binding lectin from bullfro g oocytes. To investigate the possible role of RC-RNase in the regulat ion of cell growth and differentiation during embryogenesis, its cytot oxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, b oth events were not found in RNase A-treated HeLa cells. Therefore, RC -RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNas e. (C) 1996 Academic Press, Inc.