Kinetics of Allosteric Inhibition In Vivo: a Quantitative Analysis with Synchronous Cultures of Blastocladiella emersonii.
Negative feedback control of the arginine biosynthetic pathway of Blastocladiella emersonii was observed in vivo by conducting kinetic radioisotope incorporation experiments. Transient responses of arginine precursor pools to perturbations in the medium end product concentration allowed a quantitative description of the negative feedback associated with pathway control, indicated that the N(alpha)-acetyl-l-glutamate pool size was the controlled variable, and suggested that N(alpha)-acetyl-l-glutamate and l-ornithine were the products of a transacetylation reaction in which N(alpha)-acetyl-l-ornithine and glutamic acid were the reactants. The precursors to arginine biosynthesis were found to undergo two discrete disturbances in response to altering the exogenous arginine concentration only once. An increased supply of exogenous end product resulted in diminished radioactivity in the intracellular pools of arginine, ornithine, and acetyl-glutamate, but elevated the radioisotope content of the pool of N(alpha)-acetyl-l-ornithine. Conversely, a lowered concentration of end product in the medium caused a rise in the radioisotope content of all the precursor pools except that of N(alpha)-acetyl ornithine, in which the radioactivity fell. Examination of tracer distribution among the precursors in the absence of exogenous arginine showed the nonacetylated precursor pools to be about 30 times larger than those of the acetylated intermediates and placed upper limits on the pool sizes of two pathway members that were not isolated (N(alpha)-acetyl-l-glutamic-gamma-semialdehyde and N(alpha)-acetyl-l-glutamic-gamma-semiphosphate).
Smith
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Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907.
Holmes
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