In vivo analysis of metabolic dynamics inSaccharomyces cerevisiae : I. Experimental observations

TLDR

The goal of this work was to develop a rapid sampling technique to measure transient metabolites in vivo. The authors used a rapid sampling protocol in glucose‑limited yeast cultures, adding a glucose pulse, taking 2–5 s interval samples, quenching appropriately, and measuring extracellular glucose, excreted products, glycolytic intermediates, and nucleotide cofactors by enzymatic or HPLC assays. They found that cytoplasmic and mitochondrial adenine nucleotide levels differ markedly, underscoring compartmental regulation, and that intra‑ and extracellular metabolite dynamics occur on a second‑scale, confirming rapid glycolytic regulation. © 1997 John Wiley & Sons, Inc.

Abstract

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (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 concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation 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. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305–316, 1997.