7. Collect the upper butanol phase containing
14
C-GGPP and
14 C-phytoene. Repeat the extraction, using 100 μl of butanol
and join both butanol supernatants in a single tube. The
remaining aqueous phase contains
14
C-IPP, IPP and DMAPP.
8. Add an equal volume of 1 M MgCl 2 in methanol to the butanol
sample, mix by vortexing, add another volume heptane, mix
again by vortexing. Centrifuge for 5 min at 15,000 Â g.
9. Recover the upper heptane phase containing
14 C-phytoene.
Repeat the extraction with 150 μl heptane and pool the heptane supernatants.
10. Transfer extracts into individual vials containing 6 ml of scintillation cocktail and determine the radioactivity in each fraction in a scintillation counter with an exposure time of 1 min
(see Note 11).
11. Calculation of IPP/GGPP/phytoene amounts.
(a) Empirically determine disintegrations per minute (dpm)
of 1 nmol
14 C-IPP by scintillation counting 1 μl of
14
CIPP stock in triplicate and determine the average DPM
nmol
À1 .
(b) Convert the quantified DPM of each fraction (IPP,
GGPP, phytoene) into nmol
14
C-IPP by dividing
DPM IPP /DPM GGPP /DPM phytoene by the average DPM
nmol
À1 of
14 C-IPP.
(c) Convert nmol
14 C-IPP into total nmol IPP by applying
the
14 C-IPP:IPP isotope dilution factor, for example, by
multiplication with 5.3 given the example above.
(d) Amounts of GGPP (4 molecules IPP) and phytoene
(8 molecules IPP) are then determined by dividing the
total nmol IPP in each sample by 4, and 8 respectively.
4 Notes
1. Coupling of Fractogel with Co
2+ can be performed as follows.
Shake Fractogel well to produce a slurry, remove a desired
volume for coupling and note down the volume. Remove
storage solution by filtering, transfer Fractogel into a beaker
and wash two times with Millipore water. Remove water by
filtering (avoid Fractogel to become dry). Collect Fractogel in a
beaker, add a filtered solution of 1 M CoCl 2 to fully cover
Fractogel and incubate with slow rotation for 2 h. Remove
CoCl 2 solution by filtering and wash one time with Millipore
water. Resuspend Co
2+ -coupled Fractogel in the same volume
as initially used in storage solution (20% ethanol, 150 mM
NaCl). Store at 4
C.
Enzyme Activity of Phytoene Synthase
49
14
C-GGPP and
14 C-phytoene. Repeat the extraction, using 100 μl of butanol
and join both butanol supernatants in a single tube. The
remaining aqueous phase contains
14
C-IPP, IPP and DMAPP.
8. Add an equal volume of 1 M MgCl 2 in methanol to the butanol
sample, mix by vortexing, add another volume heptane, mix
again by vortexing. Centrifuge for 5 min at 15,000 Â g.
9. Recover the upper heptane phase containing
14 C-phytoene.
Repeat the extraction with 150 μl heptane and pool the heptane supernatants.
10. Transfer extracts into individual vials containing 6 ml of scintillation cocktail and determine the radioactivity in each fraction in a scintillation counter with an exposure time of 1 min
(see Note 11).
11. Calculation of IPP/GGPP/phytoene amounts.
(a) Empirically determine disintegrations per minute (dpm)
of 1 nmol
14 C-IPP by scintillation counting 1 μl of
14
CIPP stock in triplicate and determine the average DPM
nmol
À1 .
(b) Convert the quantified DPM of each fraction (IPP,
GGPP, phytoene) into nmol
14
C-IPP by dividing
DPM IPP /DPM GGPP /DPM phytoene by the average DPM
nmol
À1 of
14 C-IPP.
(c) Convert nmol
14 C-IPP into total nmol IPP by applying
the
14 C-IPP:IPP isotope dilution factor, for example, by
multiplication with 5.3 given the example above.
(d) Amounts of GGPP (4 molecules IPP) and phytoene
(8 molecules IPP) are then determined by dividing the
total nmol IPP in each sample by 4, and 8 respectively.
4 Notes
1. Coupling of Fractogel with Co
2+ can be performed as follows.
Shake Fractogel well to produce a slurry, remove a desired
volume for coupling and note down the volume. Remove
storage solution by filtering, transfer Fractogel into a beaker
and wash two times with Millipore water. Remove water by
filtering (avoid Fractogel to become dry). Collect Fractogel in a
beaker, add a filtered solution of 1 M CoCl 2 to fully cover
Fractogel and incubate with slow rotation for 2 h. Remove
CoCl 2 solution by filtering and wash one time with Millipore
water. Resuspend Co
2+ -coupled Fractogel in the same volume
as initially used in storage solution (20% ethanol, 150 mM
NaCl). Store at 4
C.
Enzyme Activity of Phytoene Synthase
49