Thioredoxin reductase-dependent insulin disulfide reduction by phage T7 DNA polymerase reflects dissociation of the enzyme into subunits.
Phage T7 DNA polymerase contains Escherichia coli thioredoxin as a subunit and is a 1:1 complex with T7 gene 5 protein. The enzyme showed high thioredoxin activity in assays at 37 degrees C using reduction of insulin disulfides with NADPH and thioredoxin reductase, leading Randahl (Randahl, H. (1982) FEBS Lett. 150, 109-113) to propose that the thioredoxin dithiol active site is exposed in T7 DNA polymerase. However, T7 DNA polymerase and free thioredoxin differ in reactivity with iodoacetic acid after preincubation with dithiothreitol or incubation with insulin. Insulin reduction assays work at low temperatures even at 0 degrees C. The time and temperature dependence of the thioredoxin activity of T7 DNA polymerase demonstrated that dissociation into subunits at 25 or 37 degrees C accounts for the previously observed activity. Thus, T7 DNA polymerase contains the reduced form of thioredoxin with its active site SH groups masked by the subunit contact with the gene 5 protein in agreement with the results of Adler and Modrich (Adler, S., and Modrich, P. (1983) J. Biol. Chem. 258, 6956-6962). The subunit interaction of thioredoxin and gene 5 protein is salt-insensitive, but markedly temperature-dependent consistent with involvement of a hydrophobic surface area in reduced thioredoxin.
Slaby
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Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
Holmgren
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A
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*
D006146:Guanidines; D007328:Insulin; D007461:Iodoacetates; D046911:Macromolecular Substances; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D004259:DNA-Directed DNA Polymerase; D019791:Guanidine; D004229:Dithiothreitol; D019807:Iodoacetic Acid