ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 209, No. 2, July, pp. 628-636, 1981 Glutamine Synthetases from Rice: Purification and Preliminary Characterization of Two Forms in Leaves and One Form in Roots’ RAMKUMAR K. IYER, RAKESH TULI, AND JOSEPH THOMAS2 Biology and Agriculture Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India Received February 9, 1981 A relatively rapid five-step procedure was used in purifying to apparent homogeneity the glutamine synthetase from roots and one form of the enzyme (GSi) from leaves of rice. The steps were: preparation of crude extracts, ammonium sulfate precipitation, filtration on Sepharose 4B, fractionation on DEAE-Sephadex A25, and affinity chro- matography on ADP-Sepharose 4B. The purified protein appeared as a single band on polyacrylamide gel electrophoresis. Leaf GSi and the second type of leaf glutamine syn- thetase (G&I) formed distinct peaks when eluted from DEAE-Sephadex (step 4). The root enzyme and leaf GSi were similar in all the properties which were examined. Both enzymes bound to ADP-Sepharose, had similar biosynthetic (18 rmol Pi/mg protein/ min) and transferase (1324 and 1156 rmol y-glutamyl hydroxamate/mg protein/min) activities, and the same or nearly the same Km values for glutamate (2.1’7 mM), Mp (4.5 and 5.0 mM), ATP (286 MM), NH: (210 and 135 PM), and ADP (3.8 and 5.3 PM). In contrast, leaf GSn did not bind to ADP-Sepharose and had much higher Km values for glutamate (8.3 mM), Mp (15 mM), NH; (684 pM), and ADP (33 MM). Glutamine synthetase (L-glutamate: am- monia ligase (ADP forming), EC 6.3.1.2) plays a key role in the nitrogen metabo- lism of various organisms (1). The enzy- mological evidence (2-6) favoring the glu- tamine synthetase/glutamate synthase pathway (7) as the major route of am- monium assimilation in higher plants has been substantiated by tracer studies using stable (15N) and radioactive (13N) nitrogen (8-11). Glutamine synthetase extracted from whole plants (12), roots (13, 14), leaves (15,16), seeds(1’7-19), and the plant tissue of nodules (5,20) has been examined in some detail. Recently, two forms of the enzyme were identified from the leaves of rice (21) and barley (22), and from soybean hypocotyl (23). The properties and the physiological roles of the different forms of the enzyme located in different plant i This investigation formed part of a project sup- ported by the Swedish International Development Authority. ’ Author to whom correspondence and reprint re- quests should be sent. parts are not clearly understood. Purifi- cation and characterization of the en- zymes should provide further insights on their modes of function and regulation. In recent years many laboratories have developed facile procedures for the puri- fication of glutamine synthetase from mi- croorganisms (24-27). Although the en- zyme from plants has been obtained in a highly purified form in a few instances (15, 20, 28) the procedures continue to be inefficient and cumbersome. The different forms of the enzyme from plant leaves have not yet been examined in the pure state and no form of glutamine synthetase from rice has been fully purified. A com- parative assessment of the properties of the different forms of the enzyme from various parts of the same plant is of ob- vious advantage in understanding their relative significance and mode of action. In this paper we describe a relatively rapid procedure for the purification of glutamine synthetases from leaves and roots of rice and present some of their properties. 0003-9861/81/080628-09$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. 628