A study of the regulatory properties of glutamine synthetase from the nitrogen-fixing, photosynthetic bacterium, Ṟẖo̲ḏo̲p̲s̲e̲u̲ḏo̲m̲o̲ṉa̲s̲ s̲p̲ẖa̲e̲ṟo̲i̲ḏe̲s̲

Author

Houshang Karimi

Date of Award

3-1981

Degree Type

Thesis

Degree Name

Master of Science

Major Professor

K. J. Monty

Abstract

The modification of glutamine synthetase (GS) by adenylylation and the role played by this enzyme in the regulation of N₂-fixation were studied in whole cells and cell free preparations of the nitrogen-fixing, photosynthetic bacteria, Rhodopseudomonas sphaeroides.

The following evidence argued that the GS of R. sphaeroides is subject to regulation by adenylylation and deadenylylation. (a) The enzyme obtained from cells grown in the presence of ammonia was inhibited by Mg²⁺. (b) Inhibition by Mg²⁺ was relieved by exposure of the enzyme to snake venom phosphodiesterase. (c) The Mg²⁺ sensitive form of the enzyme displayed high absorbancy at 260 nm.

A study of the partially purified enzyme revealed that, upon adenylylation the enzyme is converted into a form having a more alkaline optimum pH, and a lower thermal tolerance.

The time courses of NH⁺₄-induced adenylylation of glutamine synthetase and NH⁺₄-induced inhibition of nitrogenase were studied in this microorganism to determine the degree of correlation between the two phenomena. The adenylylation of glutamine synthetase in response to NH⁺₄ addition was a rather slow process, and the extent of adenylylation depended on the concentration of ammonia added. Fast relaxation time was observed if NH⁺₄ was added at low concentrations (3μM). By contrast, the activity of nitrogenase was abolished within 8-10 seconds, the inhibition was total, and its duration depended on the ammonia concentration. The process of adenylylation can be controlled by the energy state of the cell. At high light intensity (500 J m^-2 s^-1), even in the presence of ammonia the adenylylation of glutamine synthetase tends to stay low. Ammonia was more effective in triggering the adenylylation of GS at low light intensities (130 J m^-2 s^-1). At a higher light intensity (500 J m^-2 s^-1), NH⁺₄-shock caused a slower rate of adenylylation than was seen at the lower light intensity (130 J m^-2 s^-1). The onset of the inhibition of nitrogenase by NH⁺₄, however, did not depend on the light intensity used in the experiment, the duration of inhibition of nitrogenase did not depend on the light intensity.

The question of whether glutamine synthetase was identical to the regulatory inhibitor of nitrogenase was pursued in a series of cell free experiments. There was no correlation between both the amount and the degree of adenylylation of GS in an extract and how well that extract would inhibit nitrogenase and provide a sensitivity to glutamine. These studies were further extended to a comparison of the inhibitor of nitrogenase found in crude cell extract with highly purified preparations of both forms of GS in their effect on the nitrogenase system. The purified GS was not an effective inhibitor of nitrogenase, nor did it confer upon nitrogenase a sensitivity to glutamine.

The combined studies of whole cells and cell free systems supported the conclusion that both the adenylylation of GS and the inhibition of nitrogenase result from a rising pool of glutamine and are relaxed by a fall in that pool, but that neither of those enzymes directly influence the other.

Recommended Citation

Karimi, Houshang, "A study of the regulatory properties of glutamine synthetase from the nitrogen-fixing, photosynthetic bacterium, Ṟẖo̲ḏo̲p̲s̲e̲u̲ḏo̲m̲o̲ṉa̲s̲ s̲p̲ẖa̲e̲ṟo̲i̲ḏe̲s̲. " Master's Thesis, University of Tennessee, 1981.
https://trace.tennessee.edu/utk_gradthes/15206