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The Regulation of Glutamine Metabolism in Candida utilis: Studies with 15NH3 to Measure in vivo Rates of Glutamine Synthesis
Abstract
SUMMARY: Experiments were carried out to establish whether glutamine synthesis in yeast is subject to control through the operation of cumulative feedback inhibition on the biosynthetic enzyme glutamine synthetase.
15N isotope was used to measure the in vivo rate of amide synthesis in a,culture growing on ammonia. The steady-state rate measured corresponded quite closely with a value calculated from a knowledge of the enzyme content of the yeast, and indicated that under these conditions yeast did not possess a large excess of biosynthetic capacity. In another experiment yeast was subjected to a brief period of nitrogen deprivation and rates of amide synthesis were determined immediately after addition of ammonia to the culture. This established that no significant increase in the rate of amide synthesis per unit of enzyme accompanied a reduction in the cell concentration of glutamine and other amino acids, and that the small increase in rate observed was caused by an increase in enzyme amount.
Measurements of the rate of glutamine synthesis in yeast with very high levels of enzyme confirmed that the enzyme content was of prime importance in determining the rate of synthesis in the organism. Thus when ammonia was added to yeast growing on glutamate, the rates of amide synthesis were initially nearly sixfold higher than ones observed on ammonia; this enhancement of rate almost exactly paralleled the increase in amount of enzyme in the yeast. Although the cell concentration of glutamine had greatly increased at this time and the concentration of some of its derivatives was unchanged, virtually no restraint operated to limit the synthetic activity of the enzyme in the organism. The rapid decline in the rate of glutamine synthesis which ensued occurred in response to a depletion of enzyme substrates as well as to enzyme inactivation, and the relative importance of these two factors in the control of glutamine synthesis was quantitatively assessed.
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© Society for General Microbiology, 1974