Abstract
SUMMARY: The cloned Bacteroides fragilis glutamine synthetase (GS) subunit produced in Escherichia coli had the same apparent M r of approximately 75000 as the GS subunit from B. fragilis cells. The B. fragilis GS enzyme had an apparent M r of approximately 490000 and it is concluded that the GS is a hexamer. The cloned GS did not appear to be regulated by adenylylation and deadenylylation and the cloned enzyme was inactivated by snake venom phosphodiesterase. The pH profiles of the cloned GS, assayed by the γ-glutamyl transferase (GGT) assay were similar for NH4 +-shocked and unshocked cell extracts and an isoactivity point was not obtained from these curves. The cloned GS was subject to feedback inhibition by amino acids but not by AMP. The GGT activity of the cloned GS in NH4 +-shocked and unshocked cell-free extracts was inhibited by Mg2+. Mn2+ stimulated the cloned GS GGT activity of NH4 +-shocked cell-free extracts. Western blotting indicated that GS production was regulated by nitrogen in B. fragilis cells but cell extracts showed no GGT activity. Cloned B. fragilis GS produced in E. coli was specifically and irreversibly inactivated by B. fragilis cell extracts.
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