An enzymatic cell-free assay to detect direct inhibitors of Interferon-γ

Recombinant interferon-γ (IFN-γ) from three species increases the rate of cleavage of ds-RNA by bovine seminal ribonuclease (BS-RNase), a protein with cytotoxic and antiviral activity; cleavages at the C-terminus of IFN-γ lower this activity. Here it is shown that the opposing effects of IFN-γ on the activity of two RNases, whose amino acid sequences are 81% identical, is related to their ability to bind and cleave RNA and not to contaminants in the nuclease preparations. BS-RNase and its close relative, bovine pancreatic RNase A, were secreted from Escherichia coli. The recombinant nucleases are differentially affected by EFN-γ in a manner similar to the enzymes isolated from tissues. Reaction kinetics and analysis of the degradation products by thin-layer chromatography (TLC) shows that r-BS-RNase binds RNA more tightly (saturating substrate concentration is lower) than r-RNase A. IFN-γ increases the apparent K_m of BS-RNase in proportion to V_max but has no effect on the (16-fold higher) K_m of RNase A. Heparin, which inhibits IFN-γ induction of an antiviral state and growth inhibitory effects by binding to the C-terminus of the protein, also inhibits its stimulation of BS-RNase. IFN-γ's effect on ribonuclease activity may be used to identify direct inhibitors of this multifunctional cytokine. Such assays are faster and less subject to interference from medium components than those requiring cell culture.