TABLE 1.
Levels of the sugar-derived polyols and distribution of their incorporated label generated in M. jannaschii cell extracts incubated with [6,6-2H2]glucose-6-phosphate and [U-13C]glucose-6-phosphate
| Amt of products detected and measured distribution of label with:
|
||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Controla (μM)d | [6,6-2H2]glucose-6-phosphateb
|
[U-13C]glucose-6-phosphatec
|
||||||||||||||
| μMd | 0e | 1e | 2e | 3e | 4e | μMd | 0e | 1e | 2e | 3e | 4e | 5e | 6e | 7e | ||
| Glycerol | 28f | 135 | 40.8 | 7.1 | 46.4 | 3.8 | 1.8g | 250 | 57.1 | 64.5 | 1.4 | 12.2 | 22g | |||
| d-Arabitolh | 2 | 44 | 73.5 | 4.1 | 22.4i | 11 | 44.2 | 10.5 | 15.2 | 12.2 | 10.0 | 7.9j | ||||
| Ribitolk | 6 | 120 | 82.2 | 3.9 | 13.8i | 16 | 53.9 | 11.6 | 13.2 | 10.2 | 7.0 | 4.0i | ||||
| Mannitoll (0.3) | 49 | 55.6 | 12.5 | 26.0 | 3.0 | 3.1m | 50 | 5.4 | 29.3 | 3.9 | 12.5 | 14.4 | 4.2 | 10.6 | 20m | |
| 55.8 | 10.4 | 34.6n | ||||||||||||||
| 54.8 | 10.4 | 34.6o | ||||||||||||||
| d-Glucitol | 0.3 | 180 | 55.6 | 12.5 | 26.0 | 3.0 | 3.1m | 180 | 5.9 | 36.6 | 3.8 | 11.4 | 22.3 | 1.6 | 8.3 | 10m |
| 54.8 | 10.4 | 34.6n | ||||||||||||||
| 54.8 | 10.4 | 34.6o | ||||||||||||||
| d-Altritol | ND | 7 | 48.3 | 12.9 | 21.0 | 8.8 | 9m | 7 | 5.1 | 27.8 | 4.2 | 14.6 | 13.0 | 5.1 | 10.5 | 20m |
The control sample is the analysis of a 100-μl M. jannaschii cell extract that was not incubated. All sugar assayed, except glycerol, contained one deuterium as a result of the sodium borodeuteride reduction.
In this experiment, 100 μl of M. jannaschii cell extract was incubated for 20 min at 70°C in the presence of 12 mM [6,6-2H2]glucose-6-phosphate and 4 mM (each) NADH, NADPH, ATP, GTP, UTP, TTP, and CTP prior to the analysis of sugars.
In this experiment, M. jannaschii cell extract (100 μl) was incubated for 30 min at 70°C in the presence of 7.7 mM [U-13C]glucose-6-phosphate, 3.8 mM NADH, and 3.8 mM NADPH.
Calculated from peak heights using inositol as an internal standard. ND, not detected.
The distribution of label incorporated into the polyols was calculated by adjusting the observed normalized isotopic cluster intensities of the labeled with the nonlabeled samples as previously described (2). The numbers refer to the percentage of the molecules containing the indicated number of 2H and/or 13C.
Glycerol (TMS)3 M+-15 ion at m/z 293 had 2H0 = 59%, 2H1 = 12%, and 2H2 = 29%; M+-90 ion at m/z 218 had 2H0 = 70.5%, 2H1 = 18%, and 2H2 = 4.8%; and M+-203 ion at m/z 205 had 2H0 = 80%, 2H1 = 7.2%, and 2H2 = 13%.
Measured from the M+-15 ion at m/z 293 from the (TMS)3 derivative of glycerol.
Arabitol was derived from ribulose-5-phosphate during the borohydride reduction.
Measured from the M+-90 ion at m/z 422 from the (TMS)5 derivative of ribitol and l-arabitol.
The m/z 422 ion of the arabitol (TMS)5 derivative was too weak for accurate measurement of the ion intensities. The reported values were calculated from the m/z 307 and the m/z 320 ions that showed the same labeling pattern as seen in the ribitol but with a higher abundance of 13C.
Ribitol is derived both from d-ribose-5-phosphate and d-ribulose-5-phosphate present in the cell extracts.
Mannitol is consider to arise from the borohydride reduction of the fructose-6-phosphate, fructose-1-phosphate, fructose-1,6-phosphate, and/or fructose-1,6-bisP present or generated in the cell extracts.
Measured from the M+-90-90-89 ion at m/z 345 from the (TMS)6 derivative of mannitol, glucitol, and altritol.
Measured from the m/z 307 ion from the (TMS)3 derivative of mannitol and glucitol.
Measured from the m/z 319 ion from the (TMS)6 derivative of mannitol and glucitol.