Effects of Protein RNase Inhibitor and Substrate on the Quaternary Structures of Bovine Seminal RNase

Abstract

The effect of the protein RNase inhibitor (PRI) on the activity of bovine seminal RNase (BS-RNase) was investigated using the isolated quaternary 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 but 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 cytosolic localization, also promote monomerization of the M=M form, while under identical conditions the MxM form becomes a noncovalent dimer (NCD). The sensitivity of BS-RNase or that of the isolated quaternary forms under reducing conditions thus appears to be related to differential monomerization of the two forms of the enzyme; monomer being sensitive 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 with 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 housing the PRI and the ribosomal RNA, the molecular target of the enzyme.

Second generation antitumour human RNase: significance of its structural and functional features for the mechanism of antitumour action

“…In contrast a monomeric RNase, such as RNase A (see Figure 4A), tested in the same experiment, was found to be fully inhibited in the presence of virtually stoichiometric amounts of cRI. Also in contrast, BS-RNase was found to be poorly inhibited (see Figure 4A), as previously reported [28].…”

“…Furthermore, again in contrast with the results obtained for BS-RNase [28], an extensive incubation of HHP2-RNase with a large molar excess (400-fold) of the RNase substrate UpA did not favour the formation of exchanging dimers (results not shown).…”

“…In the presence of a 2-fold molar excess of cRI over the HHP2-RNase subunit, and of increasing concentrations of UpA as a substrate, we found that cRI inhibition markedly decreased as a function of UpA concentration (see Figure 4B), so that in the presence of 1.2 mM UpA more than 80 % of HHP2-RNase molecules retained their enzymic activity. Presumably, as reported for BS-RNase [28], the interaction of substrate molecule(s) with HHP2-RNase active site(s) and\or extra site(s) hinders the formation of the enzyme-cRI complex.…”

Highly selective toxic and proapoptotic effects of two dimeric ribonucleases on thyroid cancer cells compared to the effects of doxorubicin

“…Only in malignant cells, however, the RNase is transported into the trans Golgi network, whence it is released in the cytosol (Bracale et al, 2002). The dimeric structure of BS-RNase and HHP2-RNase is essential to enter the cytosol (Murthy et al, 1996;Antignani et al, 2001;Leland et al, 2001). The cytosolic ribonuclease inhibitor (cRI) tightly binds to internalised ribonucleases, thus blocking their cytotoxic activity; only ribonucleases that can evade cRI are capable of exerting a cytotoxic action.…”

“…The cytosolic ribonuclease inhibitor (cRI) tightly binds to internalised ribonucleases, thus blocking their cytotoxic activity; only ribonucleases that can evade cRI are capable of exerting a cytotoxic action. Also, the resistance of BS-RNase to cRI inhibition depends on the dimeric structure of the enzyme (Murthy et al, 1996;Antignani et al, 2001;Leland et al, 2001). On the other hand, onconase, the frog ribonuclease, is inhibited by frog but not mammalian cRI.…”