16. Dialyze the eluate against 500 ml PSY dialysis buffer using a
standard regenerated cellulose membrane with a 15 kD cutoff,
stirring with 100 rpm in a cold room. Replace PSY dialysis
buffer with fresh buffer after 1.5 h and continue with dialysis
for another 1.5 h (see Note 8).
17. After dialysis, centrifuge reaction tubes for 20 min at
20,000 Â g (4
C), transfer supernatant into new tubes with
100 μl aliquots, immediately freeze in liquid nitrogen and store
at À80
C. Figure 2b shows steps of PSY purification as well as
purified PSY loaded on a Coomassie-stained SDS-PAGE gel
(see Note 9).
3.3 Phytoene
Synthase Activity
Assay
The following protocol includes a method on the preparation of
phosphatidylcholine liposomes according to Bozzuto and Molinari
[30]. For all subsequent steps regarding PSY activity determination, amounts are given for one measurement in a volume of 200 μl.
If kinetics are done, these amounts need to be scaled up according
to the number of time points planned. A master mix containing all
compounds is then made and samples of 200 μl volume are
removed after the desired time.
1. For the preparation of phosphatidylcholine liposomes, dissolve
20 mg phosphatidylcholine in 2 ml methanol–chloroform (1:2,
v/v) in a 15 ml reaction tube, evaporate to dryness and add
2 ml liposome buffer. Incubate for 30 min on ice, resuspend by
sonication and perform two French Press passages operated at
20,000 psi. Dilute with liposome buffer to 10 μg μl
À1 phosphatidylcholine and store at À20
C. Liposomes can be
prepared ahead and stored at À20
C for several months.
2. Prepare the reaction mix which consists of 1 μg GGPS11, an
equimolar amount of PSY, 12.5 μg phosphatidylcholine liposomes and add up to 180 μl with PSY reaction buffer. Keep the
sample on ice (see Note 10).
3. Prepare the substrate mix, consisting of 20 μM DMAPP,
16.25 μM IPP and 3.75 μM
14
C-IPP (i.e., isotope dilution of
1:5.33) and add up to 20 μl with PSY reaction buffer. Keep the
sample on ice.
4. Transfer both enzyme and substrate mix in an incubator at
20
C and preincubate for 5 min, then start the reaction by
adding the substrate to 180 μl reaction mix. Repeatedly pipet
up and down to assure that the enzymes and substrate are
mixed well.
5. Stop the reaction by adding 50 μl 160 mM EDTA and mix
thoroughly.
6. Add one assay volume butanol to the sample, vortex, and
centrifuge for 5 min at 15,000 Â g.
48
Maurizio Camagna and Ralf Welsch
standard regenerated cellulose membrane with a 15 kD cutoff,
stirring with 100 rpm in a cold room. Replace PSY dialysis
buffer with fresh buffer after 1.5 h and continue with dialysis
for another 1.5 h (see Note 8).
17. After dialysis, centrifuge reaction tubes for 20 min at
20,000 Â g (4
C), transfer supernatant into new tubes with
100 μl aliquots, immediately freeze in liquid nitrogen and store
at À80
C. Figure 2b shows steps of PSY purification as well as
purified PSY loaded on a Coomassie-stained SDS-PAGE gel
(see Note 9).
3.3 Phytoene
Synthase Activity
Assay
The following protocol includes a method on the preparation of
phosphatidylcholine liposomes according to Bozzuto and Molinari
[30]. For all subsequent steps regarding PSY activity determination, amounts are given for one measurement in a volume of 200 μl.
If kinetics are done, these amounts need to be scaled up according
to the number of time points planned. A master mix containing all
compounds is then made and samples of 200 μl volume are
removed after the desired time.
1. For the preparation of phosphatidylcholine liposomes, dissolve
20 mg phosphatidylcholine in 2 ml methanol–chloroform (1:2,
v/v) in a 15 ml reaction tube, evaporate to dryness and add
2 ml liposome buffer. Incubate for 30 min on ice, resuspend by
sonication and perform two French Press passages operated at
20,000 psi. Dilute with liposome buffer to 10 μg μl
À1 phosphatidylcholine and store at À20
C. Liposomes can be
prepared ahead and stored at À20
C for several months.
2. Prepare the reaction mix which consists of 1 μg GGPS11, an
equimolar amount of PSY, 12.5 μg phosphatidylcholine liposomes and add up to 180 μl with PSY reaction buffer. Keep the
sample on ice (see Note 10).
3. Prepare the substrate mix, consisting of 20 μM DMAPP,
16.25 μM IPP and 3.75 μM
14
C-IPP (i.e., isotope dilution of
1:5.33) and add up to 20 μl with PSY reaction buffer. Keep the
sample on ice.
4. Transfer both enzyme and substrate mix in an incubator at
20
C and preincubate for 5 min, then start the reaction by
adding the substrate to 180 μl reaction mix. Repeatedly pipet
up and down to assure that the enzymes and substrate are
mixed well.
5. Stop the reaction by adding 50 μl 160 mM EDTA and mix
thoroughly.
6. Add one assay volume butanol to the sample, vortex, and
centrifuge for 5 min at 15,000 Â g.
48
Maurizio Camagna and Ralf Welsch