Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 154

4.6.5 Development of a Cell-Free System Capable of the
Conversion of Divinyl Chlorophyllide a to Monovinyl
Chlorophyllide a
4.6.5.1 Conversion of DV Chlide a to MV Chlide a in Cucumber
Etiochloroplast
The first in organello system capable of converting DV Chlide a to MV Chlide a was
achieved in cucumber etiochloroplasts induced to accumulate DV Chlide a (Duggan
and Rebeiz 1982b ). The etiochloroplasts induced to accumulate DV Chlide a were
isolated in a medium that consisted of 0.5 M sucrose, 0.2 MTris-HCl, 1 mMMgCl 2 ,
2.5mMEDTA, 1.25 mMmethanol, 20 mMATP, 1 mMNADP and 1%BSA at a room
temperature PH of 7.7. The plastids were then subjected to a 2.5-ms pulse of actinic
white light followed by a few minutes of dark incubation during which the newly
formed DV Chlide a was converted into MV Chlide a (Duggan and Rebeiz 1982b ).
4.6.5.2 Conversion of DV Chlide a to MV Chlide a in Isolated
Cucumber Etiochloroplast Membranes
In 1995, the dependence of the reaction on NADPH was demonstrated in isolated
cucumber cotyledon etiochloroplasts (Parham and Rebeiz 1992 ).
Plastid isolation was performed under a safe green light that transmitted light
between 510 and 520 nm and which did not photoconvert DV Pchlide a to DV
Chlide a . Five g batches of DV Pchlide a -enriched cotyledons (Duggan and Rebeiz
1982a ) were hand homogenized in a cold mortar. The tissue was ground in 12.5 ml
of homogenization buffer. The latter consisted of 500 mM sucrose, 15 mM Hepes,
1 mM MgCl2, 1 mM EDTA, 9 mM Tes, 5 mM cysteine, and 0.2 % BSA (w/v),
adjusted with KOH to pH 8.0 at room temperature. The resulting homogenate was
filtered through two layers of Miracloth (Calbiochem., La Jolla, CA) and
centrifuged at 200 g for 5 min at 1 C in a Beckman JA-20 angle rotor.
The supernatant was decanted and centrifuged at 1,500 g for 10 min at 1 C. The
pelleted etiochloroplasts (about 5 mg protein) were gently resuspended with a
paintbrush, in 5.0 ml of incubation buffer. Unless otherwise indicated the latter
consisted of 500 mM Sucrose, 1.0 mM MgCl2, 2.5 mM EDTA, 20.0 mM ATP,
1.0 mM NAD+, 1.25 mM Methanol, 200.0 mM Tris and 0.2 % BSA (w/v) adjusted
to pH 7.7 at room temperature.
Preparation of plastid stroma and membranes was also performed under the safe
green light which did not photoconvert DV Pchlide a to DV Chlide a . Separation of
plastid stroma from membranes was adapted from Lee et al. ( 1991 ). Etiochlo-
roplasts (about 5 mg protein) were suspended in 5 ml of lysing buffer composed
of 1.0 mM MgCl2, 2.5 mM EDTA, 20.0 mM ATP, 1.0 mM NAD+, 1.25 mM
methanol, 0.2 % BSA (w/v), and unless otherwise indicated, 25 mM Tris-HCl.
The pH was adjusted to 7.7 at room temperature with KOH and HCl. The lysed