EFFECTS OF PROTEIN RNASE INHIBITOR AND SUBSTRATE ON THE QUATERNARY STRUCTURES OF BOVINE SEMINAL RNASE

The effect of the protein RNase inhibitor (PRI) on the activity of bov ine seminal RNase (BS-RNase) was investigated using the isolated quate rnary forms, MxM and M=M, of the enzyme reported earlier [Piccoli, R., et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1870-1874]. We found that the inhibitor does not interact with the intact isolated forms b ut has dramatic, differential effects on the two forms when the assays are performed under reducing conditions. These conditions, which are essential for full activity of the inhibitor, and are typical of its c ytosolic localization, also promote monomerization of the M=M form, wh ile under identical conditions the MxM form becomes a noncovalent dime r (NCD). The sensitivity of BS-RNase or that of the isolated quaternar y forms under reducing conditions thus appears to be related to differ ential monomerization of the two forms of the enzyme; monomer being se nsitive to PRI, The present study also shows that the interconversion between the two forms in equilibrium occurs at much higher rates in a reducing environment and that PRI further affects the interconversion and alters the equilibrium favoring monomerization of the protein. An opposite effect on the equilibrium between the forms is played by the substrate, which is found to stabilize the NCD form of the protein wit h a shift in the equilibrium between the two forms towards the dimer. These results are analyzed in the light of the antitumor action of the enzyme which is exerted in the cytosol, i.e., in the compartment hous ing the PRI and the ribosomal RNA, the molecular target of the enzyme.