Purification and some Properties of Glutamine Synthetase from the Nitrogen-fixing Cyanobacteria Anabaena cylindrica and a Nostoc sp.

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Abstract
Glutamine synthetase [EC 6.3.1.2] was purified to homogeneity from 2 N2-fixing cyanobacteria, A. cylindrica and a species of Nostoc (the phycobiont of Peltigera canina). The activities of the A. cylindrica enzyme in the biosynthetic and transferase assays were, respectively, 9.4 and 32 .mu.mol product formed min-1 (mg protein)-1; the corresponding values for the Nostoc sp. enzyme were 6.5 and 20. Stabilization of the enzyme required Mg2+, glutamate, EDTA and a thiol reagent to be present during purification. The MW of the A. cylindrica enzyme was 591,000 as estimated by sedimentation analysis, 660,000 by gel filtration and 565,000 by polyacrylamide gel electrophoresis; the Nostoc sp. enzyme gave values of 630,000 by gel filtration and 575,000 by electrophoresis. The MW of the sub-units of each enzyme were approximately 49,000-50,000. EM revealed that each molecule was composed of 12 sub-units arranged in 2 superimposed hexagonal rings. The maximum diameter of the rings was 13.6 nm and the distance between the centers of adjacent sub-units was 4.9 nm. When dialysed in the absence of stabilizing ligands the A. cylindrica enzyme lost activity and the protein band characteristic of the native enzyme was replaced by 3 bands with approximate MW of 510,000, 310,000 and 130,000. These subspecies reassociated and activity was restored by adding 2-mercaptoethanol and substrates. A similar reversible deactivation was observed with glutamine synthetase from photosynthetic eukaryotes and yeast but no similar data have been reported for a N2-fixing prokaryote.
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