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determination of Chl(ide)
a
and
b
biosynthesis in organello
.
As shown in
Table
4.4
, C, D, net Chl(ide)
a
and
b
biosynthesis was observed after 15, 30, and
60 min of incubation.
4.6 Development of Cell-Free systems Capable
of Supporting Partial Reactions of the Chlorophyll
Biosynthetic Pathway
In what follows the development of in vitro systems capable of catalyzing partial
reactions of the Chl biosynthetic pathway will be described.
4.6.1 Conversion of Protoporphyrin IX
to Mg-Protoporphyrin IX
Walker and Weinstein had described earlier a subplastidic system that overcame the
apparently mandatory requirement of plastid intactness for Mg-Proto chelatase
activity (Walker and Weinstein
1991b
). The system was prepared from lysed pea
(
Pisum sativum
) chloroplasts, and consisted of soluble and membrane-bound
fractions. Attempts at preparing similar systems from cucumber chloroplasts were
not successful (Walker and Weinstein
1991b
). Below, the preparation of a stabilized
subplastidic membrane fraction prepared from cucumber etiochloroplasts, capable
of high rates of Mg insertion into exogenous Proto, without addition of a soluble
stromal fraction, is described.
4.6.1.1 Plastid Isolation
Four-day-old etiolated cucumber cotyledons were excised with hypocotyl hooks
under subdued laboratory light (about 5 ft. candles). The excised cotyledons were
incubated at 28
C for 20 h in darkness in deep Petri dishes (80
100 mm), each
containing 3 g of tissue and 9 ml of an aqueous solution composed of 2 mM
potassium gibberellate and 0.5 mM kinetin, pH 4.3 (2, 11).
All further procedures were carried out under subdued laboratory light. After
removal of the hypocotyl hooks, 20 g of pretreated cotyledons were hand-ground in
a cold ceramic mortar containing 75 ml of homogenization medium. The latter
consisted of 500 mM sucrose, 15 mM Hepes, 30 mM Tes, 1 mM MgCl2, 1 mM
EDTA, 0.2 % (w/v) BSA, and 5 mM cysteine at a room-temperature pH of 7.7
(Lee et al.
1992
). The homogenate was filtered through two layers of Miracloth
(Calbiochem., La Jolla, CA.) and was centrifuged at 200 g for 5 min in a Beckman
JA-20 angle rotor at 1
C. The supernatant was decanted and centrifuged at 1,500 g
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