HYBRIDS FROM PEA CHLOROPLAST THIOREDOXIN-F AND THIOREDOXIN-M - PHYSICOCHEMICAL AND KINETIC CHARACTERISTICS

Two hybrid thioredoxins (Trx) have been constructed from cDNA clones c oding for pea chloroplast Trxs m and f. The splitting point was the Av ail site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli-expressed cel l lysates as a high yielding 12 kDa protein. Western blot analysis sho wed 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 Weste rn 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 purifi ed. 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 t hat with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP-malate dehydrogenase. Compu ter-assisted models of pea Trxs m and f,and of the chimeric Trx m/f, s howed a change in the orientation of the alpha(4)-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 th e regulatory cluster, and is associated with the acidic character of t his fragment and, as a consequence, with the acidic pi of the whole mo lecule. In contrast, the ability of FBPase binding and enzyme catalysi s depends on the structure on the C-side of the regulatory cysteines.