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Purification and binding features of a pea fructose‐1,6‐bisphosphatase domain involved in the interaction with thioredoxin f
Author(s) -
Hermoso Rosario,
Lázaro Juan José,
Chueca Ana,
Sahrawy Mariam,
LópezGorgé Julio
Publication year - 1999
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.1999.105421.x
Subject(s) - fructose 1,6 bisphosphatase , biochemistry , thioredoxin , fusion protein , biology , amino acid , sepharose , escherichia coli , insert (composites) , enzyme , microbiology and biotechnology , recombinant dna , gene , mechanical engineering , engineering
We previously demonstrated that a cluster in the available Asn‐ 170 Glu region of pea chloroplast fructose‐1,6‐bisphosphatase (FBPase) could be involved in its interaction with the physiological modulator thioredoxin (Trx). Using as template a cDNA coding for pea chloroplast FBPase, a DNA insert coding for a 19 amino acid fragment (Pro‐ 167 Gly) was amplified by PCR. After insertion in the pGEX‐4T vector‐1, it was expressed in Escherichia coli as a fusion protein (GST‐19) with the vector‐coded glutathione transferase (GST). This protein appears in the supernatant of cell lysates, and was purified to homogeneity. After thrombin digestion, the 19 amino acid insert was isolated as a polypeptide which displayed a positive reaction against pea chloroplast FBPase antibodies. GST‐19 linked to glutathione‐Sepharose beads, but not the GST, strongly interacts with pea Trx f , suggesting that this binding depends on the 19 amino acid insert. ELISA and Western blot experiments also demonstrate the existence of a GST‐19‐Trx f interaction, as well as a negligible quantity of Trx f bound by the vector‐coded GST. Putative competitive inhibition assays of FBPase activity carried out in the presence of increasing concentrations of the 19 amino acid insert do not demonstrate any enzyme inhibition. On the contrary, this protein fragment enhances the enzyme activity proportionally to its concentration in the assay mixture. This indicates that the FBPase‐Trx f binding promotes some type of structural modification of the Trx molecule, or of the FBPase‐Trx docking site, thus facilitating the reductive modulation of FBPase.