Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein folding, associated with isomerization of disulfide bonds, was studied using the mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) as a stable soluble and homogeneous starting material; conditions were selected to model those within the lumen of the endoplasmic reticulum where native disulfide bonds are formed in protein biosynthesis. Folding was initiated by addition of free glutathione (GSH +/- GSSG) to promote thiol-disulfide interchange and was monitored by intrinsic protein fluorescence, appearance of native ribonuclease activity, HPLC, and nonreducing SDS-PAGE. All the analyses indicated that native RNase T1 was recovered in high yield in a variety of redox conditions. Appearance of native activity followed first-order kinetics; kinetic analysis of the intrinsic fluorescence changes indicated an additional rapid process in some conditions, interpreted as the formation of a nonnative intermediate state. Analysis by HPLC and SDS-PAGE also indicated the formation of transient intermediates. In 1.5 M NaCl, GS-RNase T1 adopts a compact native-like conformation; refolding by thiol-disulfide interchange in these conditions was accelerated approximately 2-fold. Refolding of GS-RNase T1 was catalyzed by protein disulfide isomerase (PDI); substoichiometric quantities of PDI accelerated refolding several-fold. GS-RNase T1 refolding was inhibited by BiP; refolding was completely blocked in presence of a 5-fold molar excess of BiP, and the yield of refolding was substantially reduced by equimolar concentrations of BiP; the refolding was then restored by the addition of ATP. GS-RNase T1 is a convenient model substrate for studying protein folding linked to native disulfide formation in conditions comparable to those within the lumen of the endoplasmic reticulum.
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PMID:Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate. 762 8

Mixed disulfides between glutathione and the reduced forms of disulfide-bonded proteins were generated and characterized to explore their suitability as models of the unfolded state of newly-synthesized secretory proteins. RNase T1 and alpha-lactalbumin were reduced and converted to mixed disulfide derivatives, named GS-RNase T1 and GS-alpha-lactalbumin, in good yield; the molecular masses of the derivatives were confirmed by electrospray mass spectrometry. The intrinsic fluorescence of the derivatives and the binding of the hydrophobic fluorescent dye ANS were characteristic of fully unfolded proteins. Fluorescence studies and enzyme activity data indicated that GS-RNase T1 could be refolded to a nativelike state at NaCl concentrations greater than 1.5 M, as was previously demonstrated for the reduced, carboxymethylated derivative of this protein. The [NaCl]-dependent folding/unfolding equilibrium for GS-RNase T1 was reversible and could be influenced by urea. Fluorescence studies indicated that GS-alpha-lactalbumin showed a [NaCl]-dependent partial shift toward a more nativelike state, which was enhanced by the presence of Ca2+ ions. Both of the GS derivatives stimulated the ATPase activity of BiP, with apparent affinities in the range 0.1-1.0 mM. The results indicate that these GS-S-protein mixed disulfide derivatives are ideal model unfolded proteins that can be used as substrates for detailed studies on secretory protein folding in vitro and on the interactions between unfolded proteins and facilitators of protein folding.
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PMID:Protein-S-S-glutathione mixed disulfides as models of unfolded proteins. 801 32