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 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.
Murthy
B S
BS
Centre for Cellular and Molecular Biology, Hyderabad 500,007, India.
De Lorenzo
C
C
Piccoli
R
R
D'Alessio
G
G
Sirdeshmukh
R
R
D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D010926:Placental Hormones / Q000494:pharmacology*; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012661:Semen / Q000201:enzymology*; D013379:Substrate Specificity
D010926:Placental Hormones; D012329:RNA, Bacterial; C022052:placental ribonuclease inhibitor; D012260:Ribonucleases; D004229:Dithiothreitol