THIOL-DISULFIDE EXCHANGE OF RIBONUCLEASE INHIBITOR BOUND TO RIBONUCLEASE-A - EVIDENCE OF ACTIVE INHIBITOR-BOUND RIBONUCLEASE

Ribonuclease Inhibitor (RI) has been purified from pig testis. It cont ains 30 half-cystines whose oxidation affects its ability to bind and inhibit ribonuclease (RNase), By N-terminal sequence analyses testis R I showed to be identical to that from porcine liver, for which a chara cteristic all-or-none type of SH-oxidation by 5,5'-dithiobis(2-nitrobe nzoic acid) (DTNB) has been reported (Fominaya, J. M., and Hofsteenge, J. (1992) J. Biol. Chem. 257, 24655-24660), Under comparable reaction conditions, testis RI bound to RNase A did not exhibit this particula r type of oxidation; instead, bound RI got intermediate oxidation degr ees (up to 14 thiols oxidized per RI moiety) without dissociating from RNase. Moreover, RNase bound to partially oxidized RI was able to exp ress some (15%) of its potential activity (active complex). Only when DTNB treatments accounted for complex dissociation (>14 thiols oxidize d per RI moiety) the released RI molecules exhibited the all-or-none o xidation behavior. By both kinetic and circular dichroism analyses, co nformational changes have been evidenced for the transition from the i nactive to the active form of RI-RNase complex. Relaxation of RI-RNase binding without major alterations in RI structure is proposed as resp onsible for complex activation. The results are discussed in terms of a model for the reversible regulation of RNase activity mediated by th e redox status of RI.