Differential S-nitrosylation and characterization of purified S-nitrosoglutathione reductase (GSNOR) from Brassica juncea shows multiple forms of the enzyme.

Autor: Babuta P; Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi, 110007, India., Deswal R; Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi, 110007, India. Electronic address: rdeswal@botany.du.ac.in.
Jazyk: angličtina
Zdroj: Plant physiology and biochemistry : PPB [Plant Physiol Biochem] 2024 Feb; Vol. 207, pp. 108404. Date of Electronic Publication: 2024 Feb 05.
DOI: 10.1016/j.plaphy.2024.108404
Abstrakt: S-nitrosoglutathione reductase (GSNOR). a master regulator of NO homeostasis, is a single-copy gene in most plants. In Lotus japonicus, two GSNOR isoforms were identified exhibiting similar kinetic properties but differential tissue-specific expressions. Previously, a genome-wide identification in Brassica juncea revealed four copies of GSNOR, each encoding proteins that vary in subunit molecular weights and pI. Here, we report multiple forms of GSNOR using 2D immunoblot which showed 4 immunopositive spots of 41.5 kDa (pl 5.79 and 6.78) and 43 kDa (pl 6.16 and 6.23). To confirm, purification of GSNOR using anion-exchange chromatography yielded 2 distinct pools (GSNOR-A & GSNOR-B) with GSNOR activities. Subsequently, affinity-based purification resulted in 1 polypeptide from GSNOR-A and 2 polypeptides from GSNOR-B. Size exclusion-HPLC confirmed 3 GSNORs with molecular weight of 87.48 ± 2.74 KDa (GSNOR-A); 87.36 ± 3.25 and 82.74 ± 2.75 kDa (GSNOR-B). Kinetic analysis showed K m of 118 ± 11 μM and V max of 287 ± 22 nkat/mg for GSNOR-A, whereas K m of 96.4 ± 8 μM and V max of 349 ± 15 nkat/mg for GSNOR-B. S-nitrosylation and inhibition by NO showed redox regulation of all BjGSNORs. Both purified GSNORs exhibited variable denitrosylation efficiency as depicted by Biotin Switch assay. To the best of our knowledge, this is the first report confirming multiple isoforms of GSNOR in B. juncea.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Databáze: MEDLINE
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