Dissociation of Native Octameric Brain Glutamine Synthetase to a Tetramer by Treatment with N-Acetylimidazole

Native octameric glutamine synthetase from sheep brain (S_20.w = 15 S) was partially acetylated when treated with the selective reagent N-acetylimidazole, and the products formed were studied in the analytical ultracentrifuge. A two-step dissociation process was observed in which a 9S component was formed first followed by formation of a 3.8S component. Enzymatic activity declined with progressive acetylation. The 9S component could be reassociated to the 15S form by increasing ionic strength. Approximately 12 to 16 acetyl groups per subunit were introduced as determined with 1-[14C]-N-acetylimidazole. Incubation of the acetylated enzyme with hydroxylamine led to (a) reassociation of the 9S species to yield the octamer, and (b) the removal of about half of the acetyl groups. Acetylation in the presence of adenosine triphosphate (ATP) and Mg2+ did not lead to dissociation; however, subsequent removal of ATP and Mg2+ by gel filtration induced dissociation, although no 3.8S form appeared under these conditions. Addition of ATP and Mg2+ at concentrations equal to or greater than the Km for ATP in the catalytic reaction led to reassociation. The sedimentation coefficients of the products formed are in excellent agreement with expected values for tetramer (S_20.w = 9.0 S) and monomer (S_20,w = 3.7 S). The two-step dissociation process is in accord with a model possessing D₄ symmetry. The results suggest the participation of tyrosyl residues in the maintenance of the structural integrity of the enzyme and show that an additional degree of stabilization is conferred by nucleotide and metal ion. The data also indicate that the octamer is the active form of the enzyme.