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Cell organelles have been isolated from protoplast lysates and total homogenates obtained from root tips of Pisum sativum L. (cv Little Marvel) and Medicago media Pers. (cv Saranac) grown in hydroponics with nitrate nutrient solutions. Density-gradient and differential centrifugation procedures have been used to prepare mitochondria-and plastidenriched fractions in which glutamine synthetase (GS) activity was estimated. Even when purified protoplasts were gently ruptured, significant breakage of plastids occurred during preparation as shown by the high proportion of nitrite reductase recovered in the soluble fraction. Of the total GS activity recovered, up to 20% was associated with the plastid fraction, depending on the source of plant material and the GS assay utilized; when corrected for recovery of the plastid marker nitrite reductase, it was calculated that 15 to 57% of alfalfa and 14 to 64% of pea root GS was located in the plastids. A true biosynthetic assay in which glutamine production was monitored by high performance liquid chromatography was devised to estimate the physiological significance of the transferase and the semibiosynthetic assays currently used for activity measurements. When compared with the true and semibiosynthetic assays, the transferase assay for GS appeared to underestimate the root plastid enzyme. Root plastid GS was partially purified by ion-exchange chromatography, and results show that the isoenzyme found in root plastids is different from chloroplastic or cytosolic GS.In most plants, free ammonium ions are assimilated through the action of the glutamine synthetase/glutamate synthase cycle ( 17). In relation to its key role in nitrogen metabolism, GS2 has been intensively studied in leaves, roots, and nodules of higher plants (5, 10, 11, 13

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