Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic

Abstract

Ribonuclease Sa (pI = 3.5) from Streptomyces aureofaciens and its 3K (D1K, D17K, E41K) (pI = 6.4) and 5K (3K + D25K, E74K) (pI = 10.2) mutants were tested for cytotoxicity. The 5K mutant was cytotoxic to normal and v-ras-transformed NIH3T3 mouse fibroblasts, but RNase Sa and 3K were not. The structure, stability, and activity of the three proteins are comparable, but the net charge at pH 7 increases from -7 for RNase Sa to -1 for 3K and to +3 for 5K. These results suggest that a net positive charge is a key determinant of ribonuclease cytotoxicity. The cytotoxic 5K mutant preferentially attacks v-ras-NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras-oncogene are potential targets for ribonuclease-based drugs.

Fig. 1.

(A) Viability of normal NIH3T3 fibroblasts (blank columns) and v-ras-NIH3T3 fibroblasts (hatched columns) treated for 24 h with RNase Sa and its 3K and 5K mutants at a concentration of 500 μg/mL. Values are expressed as percent viability of control cells grown without RNase. (B,C) Effect of 5K RNase Sa on the cell number (B) and respiration rate (C) of normal NIH3T3 fibroblasts (blank columns) and v-ras-NIH3T3 fibroblasts (hatched columns) treated for 24 h with various concentrations of 5K RNase Sa: bars a and e, untreated; b and f, 10 μg/mL; c and g, 100 μg/mL; and d and h, 500 μg/mL.