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 contains 30 half-cystines whose oxidation affects its ability to bind and inhibit ribonuclease (RNase). By N-terminal sequence analyses testis RI showed to be identical to that from porcine liver, for which a characteristic all-or-none type of SH-oxidation by 5,5'-dithiobis(2-nitrobenzoic 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 particular type of oxidation; instead, bound RI got intermediate oxidation degrees (up to 14 thiols oxidized per RI moiety) without dissociating from RNase. Moreover, RNase bound to partially oxidized RI was able to express some (15%) of its potential activity (active complex). Only when DTNB treatments accounted for complex dissociation (> 14 thiols oxidized per RI moiety) the released RI molecules exhibited the all-or-none oxidation behavior. By both kinetic and circular dichroism analyses, conformational changes have been evidenced for the transition from the inactive to the active form of RI-RNase complex. Relaxation of RI-RNase binding without major alterations in RI structure is proposed as responsible for complex activation. The results are discussed in terms of a model for the reversible regulation of RNase activity mediated by the redox status of RI.
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013552:Swine; D013737:Testis / Q000378:metabolism
D004220:Disulfides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D013438:Sulfhydryl Compounds; C066953:ribonuclease inhibitor, porcine; D004228:Dithionitrobenzoic Acid; D012259:Ribonuclease, Pancreatic