IN-VIVO ANALYSIS OF METABOLIC DYNAMICS IN SACCHAROMYCES-CEREVISIAE .1. EXPERIMENTAL-OBSERVATIONS

The goal of this work was to obtain rapid sampling technique to measur e transient metabolites in vivo. First, a pulse of glucose was added t o a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s inter vals and quenched using methods that depend on the metabolite measured . Extracellular glucose, excreted products, as well as glycolytic inte rmediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods . Significant differences between the adenine nucleotide concentration s in the cytoplasm and mitochondria indicated the importance of compar tmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (C) 1997 John Wiley & Sons, Inc.