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

M Dragicevic,Ana Simonovic,Sladjana Todorovic,Danijela Misic,Vanja Tanackovic,Milica Bogdanovic,Tijana Cvetic

doi:10.2298/abs1104965d

M Dragicevic, Ana Simonovic + Show 5 more

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https://doi.org/10.2298/abs1104965d

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Abstract

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.

Highlights

Glutamine synthetase (GS, EC 6.3.1.2) facilitates the assimilation of inorganic nitrogen in the form of ammonia with glutamate, forming glutamine
Spinacia oleracea has been classified as a plant expressing only GS2 activity in leaves (Hirel et al, 1982, 1984; McNally et al, 1983; Ericson, 1985), even though both GS1 and GS2 sequences have been deposited to the GenBank (EU057984 and EF143582)
Hereinwith we present high-resolution spinach GS zymograms: one GS1 expressed in the roots and two distinct GS2 activity bands

Summary

Introduction

Glutamine synthetase (GS, EC 6.3.1.2) facilitates the assimilation of inorganic nitrogen in the form of ammonia with glutamate, forming glutamine. The developed activity bands were photographed and cut out from the gel, equilibrated in 50 mM Tris-HCl pH 8 for one hour at room temperature, homogenized by the syringe maceration method (Scheer et al, 2001) and extracted for one hour at 500C with 200 μl buffer containing 50 mM Tris HCl pH 8, 1% SDS and 3% β-mercaptoethanol.

Results

Conclusion