Add time:09/09/2019         Source:sciencedirect.com

A modification of the method of Kauffman et al. (F. C. Kauffman, J. G. Brown, J. V. Passonneau, and O. H. Lowry (1969)J. Biol. Chem.244, 3647–3653) for the spectrophotometric determination of xylulose 5-phosphate, ribulose 5-phosphate, and combined ribose 5-phosphate and sedoheptulose 7-phosphate in tissue extract is presented. Using commercially available enzymes all three assays come to a clear endpoint with the assays described. Values for these metabolites in liver in three dietary states are reported; 48 h starved, ad libitum feeding of standard NIH rat ration, and meal feeding of a fat-free diet. Xylulose 5-phosphate values were 3.8 ± 0.3, 8.6 ± 0.3, and 66.3 ± 8.3 nmol/g. Ribulose 5-phosphate values were 3.4 ± 0.3, 5.8 ± 0.2, and 37.1 ± 5.3 nmol/g. Combined ribose 5-phosphate and sedoheptulose 7-phosphate were 29.3 ± 0.3, 38.2 ± 1.2, and 108.2 ± 14.5 nmol/g. The ratio of measured tissue content of [xylulose 5-phosphate]/[ribulose 5-phosphate] was found to be 1.12 ± 0.07 in starved animals, 1.48 ± 0.04 in ad libitum fed animals and 1.78 ± 0.03 in low-fat meal fed animals. These data are in good agreement with the range of equilibrium constants reported for this reaction, suggesting that the ribulose 5-phosphate 3-epimerase reaction (EC 5.1.3.1) is a near equilibrium reaction despite a more than 10-fold change in the tissue content of these metabolites.

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