Activation of partially folded mitochondrial malate dehydrogenase by thioredoxin.
CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenase is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.
Li
W
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Department of Biochemistry & Cellular & Molecular Biology, University of Tennessee, Knoxville, USA.
Churchich
J E
JE
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013552:Swine; D013879:Thioredoxins / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006146:Guanidines; D013879:Thioredoxins; C027132:1-anilino-8-naphthalenesulfonate; D004228:Dithionitrobenzoic Acid; D008291:Malate Dehydrogenase; D019791:Guanidine; D004229:Dithiothreitol