Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics.
Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the Avall 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 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 the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pl 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.
Department of Plant Biochemistry, Estación Experimental del Zaidín (CSIC), Granada, Spain.
J
J
López Jaramillo
A
A
Chueca
M
M
Sahrawy
J
J
López Gorgé
D000595:Amino Acid Sequence; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004355:Drug Stability; D004926:Escherichia coli; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D054479:Chloroplast Thioredoxins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013879:Thioredoxins