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Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics
- Jaramillo, J.L., Chueca, A., Sahrawy, M., Lopez-Gorge, J.
- The plant journal 1998 v.15 no.2 pp. 155-163
- Escherichia coli, biosynthesis, complementary DNA, immunochemistry, heat stability, fructose-bisphosphatase, recombinant proteins, isoelectric point, amino acid sequences, malate dehydrogenase, Pisum sativum, molecular weight, molecular conformation, cysteine, chloroplasts, plant proteins
- Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the AvaII site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli-expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose-1,6-bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP-malate dehydrogenase. Computer-assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the alpha4-helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N-side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pI of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C-side of the regulatory cysteines.