Isolation and functional characterization of two thioredoxin h isoforms from grape.

Thioredoxins (Trxs) are small ubiquitous proteins that participate in dithiol-disulfide exchange reactions. In contrast to animals and prokaryotes, plants possess different types of Trxs that play a vital role in a number of different cellular processes. Two full-length cDNAs encoding different Trx h isoforms, designated VvTrx h2 and VvTrx h3, were isolated and cloned from grape (Vitis vinifera L. cv. Askari) berry tissue by rapid amplification of cDNA ends (RACE) method. VvTrx h2 and VvTrx h3 were heterologously expressed in Escherichia coli and their activities were compared using DTT-dependent insulin reduction and 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) reduction activities. The NADPH-dependent DTNB reduction assay demonstrated that the both VvTrx h isoforms were reduced by NADPH-dependent thioredoxin reductase (NTR) from E. coli. Under heat shock treatment, the recombinant VvTrx h proteins formed the oligomeric structures at above 50 °C with a decrease in their disulfide reductase activities. The redox-dependent structural changes of VvTrx h2 and VvTrx h3 revealed that their oligomeric structures were changed into monomers and significantly increased their disulfide reductase activities. Furthermore, the both recombinant proteins were able to conserve a DTNB reduction activity even after 15 min heating at 99 °C.