Single-Run Separation and Detection of Multiple Metabolic Intermediates by Anion-Exchange High-Performance Liquid Chromatography and Application to Cell Pool Extracts Prepared fromEscherichia coli
✍ Scribed by Meenakshi Bhattacharya; Lorra Fuhrman; Andrew Ingram; Kenneth W. Nickerson; Tyrrell Conway
- Publisher
- Elsevier Science
- Year
- 1995
- Tongue
- English
- Weight
- 121 KB
- Volume
- 232
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A method is described for analysis of the intracelluis shown in the following examples. One approach for lar concentrations of metabolic intermediates of the applying metabolic control theory to the study of flux Embden-Meyerhof -Parnas pathway, the Entnerthrough metabolic pathways requires knowledge of me-Doudoroff pathway, the pentose phosphate pathway, tabolite pool sizes (1,2). In fact, experimental studies and the tricarboxylic acid cycle in cell pool extracts of metabolic control theory point out that pathway flux of Escherichia coli. A single anion-exchange HPLC can be extraordinarily sensitive to changes in certain run of 40 min allowed resolution of 27 anionic metabometabolite concentrations (3-6). The in vitro analysis lite standards. Detection limits of 0.1 nmol per injecof metabolite transfer via enzyme-enzyme complexes tion were achieved by use of a conductivity detector (7) is being applied to in vivo studies of metabolite equipped with an anion self-regenerating suppressor channeling through multienzyme complexes (8). For and a uv detector. A boiling water extraction proceexample, investigations of channeling of TCA 2 cycle dure was used to prepare cell pool extracts. Cochrometabolites in Saccharomyces cerevisiae (9) and chanmatography of cell pool extracts and metabolite neling of metabolites through nucleotide biosynthetic standards was used to confirm the identities of metabpathways to the DNA replication machinery of bacteolites in the cell pool. As many as 16 metabolites could riophage T4-infected Escherichia coli (10) require mulbe detected and quantified in the cell pool extracts tiple experimental approaches, including measurement by using the described HPLC method. An analysis of tabolite accumulations led to introduction of a fourth