RESUMEN
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 15â¯min heating at 99⯰C.
Asunto(s)
Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Tiorredoxina h/aislamiento & purificación , Tiorredoxina h/metabolismo , Vitis , Biocatálisis , Clonación Molecular , Respuesta al Choque Térmico , Insulina/metabolismo , Oxidación-Reducción , Proteínas de Plantas/química , Proteínas de Plantas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Estabilidad Proteica , Análisis de Secuencia , Temperatura , Tiorredoxina h/química , Tiorredoxina h/genéticaRESUMEN
Thioredoxins (Trxs) play important roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. They execute their function by regulating the oxidation and reduction of disulfide bonds. ACHT1 (atypical cysteine/histidine-rich Trx1) is a thylakoid-associated thioredoxin-type protein found in the Arabidopsis thaliana chloroplast. Recombinant ACHT1 protein was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal diffracted to 1.7â Å resolution and a complete X-ray data set was collected. Preliminary crystallographic analysis suggested that the crystals belonged to space group C2221, with unit-cell parameters a = 102.7, b = 100.6, c = 92.8â Å.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Cloroplastos/química , Tiorredoxina h/química , Secuencia de Aminoácidos , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Cloroplastos/enzimología , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Oxidación-Reducción , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Tiorredoxina h/genética , Tiorredoxina h/aislamiento & purificación , Difracción de Rayos XRESUMEN
The cDNA code of thioredoxin h, designated as HbTRX1, was isolated from Hevea brasiliensis by rapid amplification of cDNA ends. HbTRX1 contained a 542-bp open reading frame encoding 123 amino acids. The deduced HbTRX1 protein showing high identity to thioredoxin h of other plant species was predicted to possess the conserved catalytic site WCXPC. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that HbTRX1 was constitutively expressed in all tested tissues. HbTRX1 transcripts accumulated at relatively low levels in the flower, somatic embryo, and leaves, while HbTRX1 transcripts accumulated at relatively high levels in the callus and latex. The HbTRX1 transcript was expressed at different levels, with higher levels in self-rooting juvenile clones than in their donor clones. HbTRX1 was expressed in Escherichia coli, and its activity was demonstrated using the dithiothreitol-dependent insulin assay. This work provides a basis for studying the biological function of thioredoxin h in rubber tree.