Isolation and characterization of an extended thioredoxin h from poplar

Physiologia Plantarum - Tập 114 Số 2 - Trang 165-171 - 2002
Éric Gelhaye1, Nicolas Rouhier2, Pascal Laurent2, P. Sautière3, Francis Martin4, Jean‐Pierre Jacquot2
1Institut National de la Recherche Agronomique, Rabat, Morocco
2UMR IaM 1136 INRA‐Université H. Poincaré‐Nancy I. Faculté des Sciences F‐54506 Vandoeuvre, France
3Laboratoire d'Endocrinologie des Annélides UPRESA 8017. Université des Sciences et Technologies de Lille, F‐59655 Villeneuve d'Ascq Cedex, France
4UMR IaM 1136 INRA‐Université H. Poincaré‐Nancy I. Centre INRA de Nancy., F‐54280 Champenoux, France

Tóm tắt

A cDNA coding for a thioredoxin h has been isolated from a xylem/phloem poplar cDNA library by RACE‐PCR. The nucleotide sequence called popTrx‐h2 is homologous to other thioredoxins h isolated from plants but differs from the other thioredoxins h by presenting a 30 amino acid long N‐terminus extension. A variant of this cDNA lacking the N‐terminal extension was also generated by PCR. Both cDNAs have been introduced into an expression plasmid (pET‐3d) and the recombinant proteins have been expressed to a high level and purified from Escherichia coli cells. Protein sequencing showed that a part of the N‐terminal extension was cleaved in the E. coli cells, with the first 19 amino acids missing, suggesting the presence of a putative cleavage site in the N‐terminal extension of popTrx‐h2. Both recombinant proteins display unusual catalytic properties compared to other thioredoxins h characterized so far, i.e. a weak reduction by Arabidopsis thaliana NADPH‐dependent thioredoxin reductase, and a weak activation of the chloroplastic NADP‐malate dehydrogenase, a non‐physiological target enzyme. Northern blot experiments indicate that the transcripts of popTrx‐h2 are present in leaves and roots, albeit at a lower level compared to the earlier characterized popTrx‐h1.

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Tài liệu tham khảo

10.1006/jmbi.1990.9999

10.1016/S0981-9428(00)00760-9

10.1105/tpc.8.9.1641

10.1006/jmbi.2000.4006

10.1016/0003-9861(88)90282-2

10.1046/j.1432-1327.1998.2520314.x

10.1021/bi982783v

10.1111/j.1432-1033.1968.tb00470.x

Holmgren A, 1979, Reduction of disulfides by thioredoxin. Exceptional reactivity of insulin and suggested functions of thioredoxin in mechanism of hormone action, J Biol Chem, 254, 9627, 10.1016/S0021-9258(19)86818-7

10.1006/bbrc.1998.9139

10.1111/j.1432-1033.1991.tb16090.x

10.1016/0076-6879(95)52027-9

10.1046/j.1469-8137.1997.00784.x

10.1016/S0969-2126(94)00086-7

10.1021/bi9817818

10.1016/0022-2836(90)90313-B

10.1016/S0378-1119(99)00190-0

10.1016/S0167-4838(99)00235-6

10.1073/pnas.92.12.5620

10.1104/pp.010586

SambrookJ FrischEF ManiatisT(1989) Molecular cloning: A laboratory manual.Cold Spring Harbor New York NY

10.1146/annurev.arplant.51.1.371

10.1007/BF00020396

10.1016/0022-2836(91)90855-Z

10.1074/jbc.274.28.19714

10.1111/j.1432-1033.1992.tb17331.x

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Thông tin xuất bản

Physiologia Plantarum

Tập 114 Số 2

165-171

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