PURIFICATION AND INITIAL STUDIES ON THE REGULATION OF GLUTAMINE SYNTHETASE IN AGMENELLUM QUADRUPLICATUM.

摘要

A rapid permeabilized whole cell assay was developed for assaying glutamine synthetase from the blue-green alga Agmenellum quadruplicatum strain PR-6. This procedure involved the use of the non-ionic detergent Nonidet P-40 to allow the diffusion of substrates and products through the cell membrane. The concentrations of the assay components were 10 mM ATP, 20 mM MgC(,2), 20 mM NH(,4)Cl, 50 mM glutamate, 100 mMHepes (pH 7.6), and 0.5% Nonidet P-40. The assay mixture was incubated at 39(DEGREES)C for 10 minutes.;In cells grown on nitrate, an increase in glutamine synthetase activity per mg cell dry weight from 31.6 to 47.9 was observed from 18 to 25 h. An increase in activity from 5.5 to 29.5 units/mg cell dry weight was observed in cells grown on ammonia between 12.5 and 16 h; and in cells grown on urea, the increase in glutamine activity was from 20.9 to 50.1 units/mg cell dry weight between 17 and 20.5 h.;During the transition from nitrogen replete to nitrogen depleted growth, the C-phycocyanin and nitrogen content of the cells were monitored. In cells grown on nitrate and ammonia, the decline in both of these parameters coincided with the depletion of nitrogen from the medium, the end of exponential growth, and the increase in glutamine synthetase activity per cell dry weight. The decline of C-phycocyanin and nitrogen content in the cells grown on urea began at the same time, but preceded urea depletion by 15 h. These data provided information for optimizing cellular content of glutamine synthetase for enzyme purification. Moreover, these data strongly suggest nitrogen catabolite repression of glutamine synthetase in A. quadruplicatum.;Glutamine synthetase was purified by a combination of centrifugation, affinity chromatography on Sepharose 4B-anthranilic acid resin, and DE-52 ion-exchange resin. A 328-fold purification of the enzyme was achieved with 21.9% recovery using this procedure. The native molecular weight of the purified protein as determined by gel electrophoresis was 546,000 and 577,000 by sedimentation-equilibrium ultracentrifugation. From sodium dodecyl sulfate gel electrophoresis the subunit molecular weight was calculated to be 55,300. A study of electron micrographs showed the native protein to consist of two six-membered hexagonal rings. The apparent Michaelis constants for glutamate, ammonia and ATP (constant Mg('++): ATP ratio of 2.0) were 5.2 mM, 0.097 mM, and 2.8 mM, respectively. The physical properties of glutamine synthetase indicate a similarity to the glutamine synthetase of bacteria rather than to the glutamine synthetase of eucaryotic cells.;This assay was used to monitor glutamine synthetase activity during growth of A. quadruplicatum on a limiting concentration of nitrate, ammonia, or urea. In cultures of A. quadruplicatum grown on nitrate, total glutamine synthetase activity increased exponentially during the entire time course of the experiment, while in cells grown on ammonia the increase in total activity was not observed until ammonia was depleted from the medium. Glutamine synthetase activity in A. quadruplicatum grown on urea began to increase 8 h prior to urea depletion from the medium.