Purification and some properties of glutamine synthetases from bifidobacteria.

Abstract

Glutamine synthetases [l-glutamate; ammonia ligase (ADP forming), EC 6.3.1.2] were purified, respectively, 570-, 220- and 80-fold with 12, 5 and 0.5% yields from cell-free extracts of Bifidobacterium bifidum a, B. breve 203 and B. pseudolongum a. The purification procedure consisted of cell disruption, ammonium sulfate fractionation and column chromatographies on DEAE- cellulose, Sepharose 6B and hydroxylapatite. The purified enzymes were judged to be homogeneous on polyacrylamide disc gel electrophoresis. They had similar molecular weights of about 500,000. The optimum pH was 6.0 ~ 6.5 for their biosynthetic reactions with 30 mm Mg2 +, and 6.5 for their γ-glutamylhydroxamate forming (transferring) reactions. The Km values for l-glutamate, ammonia and ATP in their biosynthetic reactions were, respectively, 18~25mm, 0.56 ~ 0.69mm and 1.5 ~ 2.0mm with 30 mm Mg2+ as a cofactor. When 7.5 mm Mn2+ was substituted for 30 mm Mg2 +, the Km for l-glutamate decreased by 8.6 ~23-fold. The optimum pH for the biosynthe...