MECHANISM OF RIBONUCLEASE CYTOTOXICITY

Bovine seminal ribonuclease (BS-RNase), a dimeric homolog of bovine pa ncreatic ribonuclease A (RNase A), is toxic to mammalian cells. In con trast to dimeric BS-RNase, monomeric BS-RNase and RNase A are not cyto toxic and are bound tightly by cytosolic ribonuclease inhibitor. To el ucidate the mechanism of ribonuclease cytotoxicity, we constructed a s eries of hybrid and semisynthetic enzymes and examined their propertie s. In five hybrid enzymes, divergent residues in BS-RNase were replace d with the analogous residues of RNase A so as to diminish an interact ion with a putative cellular receptor, In a semisynthetic enzyme, the disulfide bonds that cross-link the monomeric subunits of dimeric BS-R Nase were replaced with thioether bonds, which can withstand the reduc ing environment of the cytosol. Each hybrid and semisynthetic enzyme h ad ribonucleolytic and cytotoxic activities comparable with those of w ild-type BS-RNase. These results suggest that dimeric BS-RNase (pI = 1 0.3) enters cells by adsorptive rather than receptor-mediated endocyto sis and then evades cytosolic ribonuclease inhibitor so as to degrade cellular RNA. This mechanism accounts for the need for a cyto solic ri bonuclease inhibitor and for the cytotoxicity of other homologs of RNa se A.