13C n.m.r. isotopomer and computer-simulation studies of the non-oxidative pentose phosphate pathway of human erythrocytes

Abstract

13C double-quantum filtered correlation spectroscopy (DQF-COSY) provides a novel method for the detection of reactions involving carbon-bond scissions. We report the use of this technique to investigate isotopic exchange reactions of the non-oxidative pentose phosphate pathway in human erythrocytes. These exchange reactions resulted in the formation of a range of isotopic isomers (isotopomers) of glucose 6-phosphate after incubation of a mixture of universally 13C-labelled and unlabelled glucose 6-phosphate with fructose 1,6-bisphosphate and haemolysates. These isotopomers were detected in the coupling patterns of cross-peaks within the DQF-COSY spectrum of the deproteinized sample. A computer model which fully describes the reactions of the non-oxidative pentose phosphate pathway in human erythrocytes has previously been constructed and tested with 31P n.m.r. time-course data in our laboratory. This model was refined using 13C n.m.r. time-course data and extended to include the range of isotopomers which may be formed experimentally by the reactions of the non-oxidative pentose phosphate pathway. The isotopomer ratios obtained experimentally from the DQF-COSY spectrum were consistent with simulations generated by this model.

Demonstration of glucose‐6‐phosphate hydrogen 5 enrichment from deuterated water by transaldolase‐mediated exchange alone

“…Hemolysates were incubated at 37 C for 45 min before their addition to metabolic substrates to deplete reducing equivalents and ATP (12). G6P enriched to 5% with [U- 13 C]G6P (95 mmol) and F-1,6-P 2 (57 mmol) were dissolved in 1 mL TRIS buffer (TRIS, 50 mM; KCl, 90 mM; NaCl, 10 mM; MgCl 2 2 mM; DL-dithiothreitol,1 mM; ethylenediaminetetraacetic acid, 0.5 mM; 10% 2 H 2 O, pH 7.4).…”

Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices

“…NMR spectroscopy has recently been used for the determination of metabolic flux distributions in bacteria (Inbar and Lapidot, 1987;Marx et al, 1996;Walsh and Koshland, 1984), mammalian cell cultures (Portais et al, 1993;Sharfstein et al, 1993;Stephanopoulos, 1994, 1995), erythrocytes (Berthon et al, 1993), and organs (Chance et al, 1983;Fernandez and Des Rosiers, 1995;Malloy et al, 1988Malloy et al, , 1990, as well as in characterization of carbohydrate metabolism of humans (Gopher et al, 1990). Measurements of the label distribution after feeding 13 C-labeled substrates has been used to calculate flux ratios at branch points of metabolic networks, on top of the conventional measurements of extracellular fluxes for metabolic flux analysis (Marx et al, 1996).…”

Revisiting the 13C‐label distribution of the non‐oxidative branch of the pentose phosphate pathway based upon kinetic and genetic evidence

“…1. Whereas the correct ping-pong mechanism for TK and TA was adopted by several researchers in the 1990s to construct detailed kinetic models [20,[26][27][28], this has been largely overlooked by the metabolic engineering community.…”

“…The six additional reactions consist of five stoichiometric neutral reactions, two of which are catalyzed by TA (r.8 and r.9) and three of which are catalyzed by TK (r.5, r.6 and r.7), and one additional reversible TK-catalyzed reaction (r.4). Although the stoichiometric neutral reactions have no effect on the mass balances set up over the system, they do influence the labeling pattern of the metabolite pools and thus need to be incorporated into the metabolic network for 13 C-based flux estimations [18,20]. The structure of reactions r.1-9 is such that a carbon fragment is transferred from one substrate to another, yielding two products.…”