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The enzyme catalyzing the final step of ammonia assimi lation, glutamate synthase (EC 1.4.7.1), has been partially purified, and its occurrence confirmed in a number of microorganisms, Escherichia coli,1,2) Klebsiella aerogenes,3) Bacillus megaterium,4) yeast,5) Thiobacilllus thioparus,6) Caulobacter crescentus,7) Rhodopseudomonas capsulata,8) Nostoc ellipsosporum,9) Anabaena cylindrica9) and Chlorella fusca10) as well as in several higher plants11) including Vicia faba.12) Although the enzyme has been located in a variety of organisms, extensive purification has only been accom plished in the case of E. coli, K. aerogenes and B. megaterium. In this communication, we wish to report on the purification and some properties of ferredoxin dependent glutamate synthase from spinach leaves. Ferredoxin was purified from spinach leaves as described by Tagawa and Arnon13) (A422: A277=0.43). The enzyma tic assay was based on the initial rate of oxidation of zinc-reduced methyl viologen following the addition of 2 oxoglutarate. In a Thunberg-type cuvette (light path, 1 cm) were added l0ƒÊmol of glutamine, 500ƒÊmol of Tricine KOH buffer, pH 7.5, 100ƒÊmol of potassium phosphate buffer, pH 7.5, 3 mg of bovine serum albumin, 1 mg of ferredoxin and the enzyme to a total volume of 1.79 ml. The cell was made anaerobic by repeated evacuation and flushing with argon. Then a constant volume (0.61 ml) of reduced methyl viologen (0.67 ƒÊmol) was anaerobically introduced into the evacuated cuvette with the apparatus of Asada et al.14) After an initial incubation for 5 min to permit measurement of endogenous bleaching and to attain a temperature equilibrium (25•Ž), the reaction was initiated by adding 0.1 ml of 2-oxoglutarate (10 ƒÊmol) from the side arm. The rate of oxidation of reduced methyl viologen was calculated from the change in absorbancy at 604nm after the addition of 2-oxoglutarate. Absorbancy was measured with a Hitachi 556 recording spectrophotometer. A unit of glutamate synthase was defined as the amount of enzyme which consumed I ƒÊmol of reduced methyl viologen per minute under the conditions described. To obtain a crude extract, spinach leaves were homogenized in three volumes of 50 mm potassium phosphate buffer, pH 7.2, containing 10 mM (ƒÀ-mercaptoethanol, 5 mM EDTA and I mM PMSF (phenylmethyl sulfonyl fluoride), in a Waring blender.

The purification and properties of glutamate synthase from spinach leaves, and its dependence on ferredoxin.