Coupling native page/activity-staining with SDS-PAGE/immunodetection for the analysis of glutamine synthetase isoforms in spinach | Zendy

Milan Dragićević | Zendy; Vanja Tanackovic | Zendy; Danijela Mišić | Zendy; Tijana Cvetić Antić | Zendy; Slađana Todorović | Zendy; Milica Bogdanović﻿﻿﻿ | Zendy; Ana Simonović | Zendy

Open Access

Coupling native page/activity-staining with SDS-PAGE/immunodetection for the analysis of glutamine synthetase isoforms in spinach

Author(s) -

Milan Dragićević,

Vanja Tanackovic,

Danijela Mišić,

Tijana Cvetić Antić,

Slađana Todorović,

Milica Bogdanović,

Ana Simonović

Publication year - 2011

Publication title -

archives of biological sciences

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.217

H-Index - 25

eISSN - 1821-4339

pISSN - 0354-4664

DOI - 10.2298/abs1104965d

Subject(s) - spinach , spinacia , glutamine synthetase , polyacrylamide gel electrophoresis , gene isoform , biochemistry , isozyme , gel electrophoresis , enzyme , biology , glufosinate , cytosol , microbiology and biotechnology , glutamine , chloroplast , amino acid , gene , glyphosate , agronomy

Glutamine synthetase (GS) is a key nitrogen-assimilating enzyme in plants and a target for the broad-spectrum herbicide glufosinate. Understanding its kinetic and structural properties is of major agricultural importance. Spinach (Spinacia oleracea) is classified as a plant expressing only chloroplastic GS activity. We have analyzed soluble proteins in the spinach by coupling native polyacrylamide gel electrophoresis (PAGE)-activity detection, based on phosphate precipitation, with SDS-PAGE/immunoblotting. One cytosolic (GS1) isoform from the roots and two chloroplastic (GS2) isoforms expressed in leaves were resolved by native PAGE. The identity of the obtained bands was established by the application of GS-specific inhibitors, L-methionine sulfoximine and glufosinate. Examination by sodium dodecyl sulfate (SDS)-PAGE/ Western analysis with anti-GS antibodies, confirmed the identity of the active bands and revealed that both chloroplastic isoforms are composed of 44 kDa subunits, while the cytosolic isoform consists of 40 kDa subunits. The presence of more GS2 isozymes than encoded in the spinach genome is discussed in terms of posttranslational modifications

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