Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 144

centrifuge tube and centrifugation at 6,000 g for 5 min in a Beckman JS-13

swinging bucket rotor at 1 C. Intact plastids recovered as a pellet were gently
resuspended in 5 cm 3 of medium consisting of 0.5 M sucrose, 0.2 M Tris-HCl,
20 mM MgCl2, 2.5 mM EDTA, 1.25 mM methanol, 20 mM ATP, 40 mM NAD,
8 mM methionine, and 5

μ

M phytol at a room temperature, pH 7.7 (Daniell and
Rebeiz 1982b ; Rebeiz et al. 1984 ).
Etioplast incubations consisted of 0.95 ml of plastid suspension (3-5 mg protein)
and 0.05 ml of 10 mMALA. Incubation was carried out at 28 C for 15-60 min on a
reciprocating water bath operated at 50 oscillation per min under 4 umol/m 2 /s of
cool white fluorescent light. Incubations were terminated by precipitation with
10 ml of acetone: 0.1 M NH 4 OH (9:1, v/v). The acetone extracts containing the
tetrapyrrole pigments were cleared of insoluble lipoproteins by centrifugation at
39,000 g for 12 min. Chl a, a fully esterified tetrapyrrole, was removed from the

aqueous acetone solution by extraction with 1 volume of hexane followed by a
second extraction with 1/3 volume of hexane. The more polar monocarboxylic
tetrapyrroles such as Pchlide a and Chlide a remained in the hexane-extracted
aqueous acetone fraction. The amount of Pchlide a and Chlide a was determined
spectrofluorometrically on aliquots of the hexane-extracted acetone fraction as
described in (Rebeiz et al. 1975a ). One ml aliquot of the hexane extract containing
the Chl was dried under N 2 gas and the residue was redissolved in 4 ml of 80 %
acetone. The amount of Chl a and b in the acetone solution was determined
spectrofluorometrically as described in (Bazzaz and Rebeiz 1979 ) Fluorescence
spectra were recorded as described in (Rebeiz et al. 1975a ) . The endogenous ALA
content of the isolated plastids was determined as described by Mauzerall and
Granick ( 1956 ).

4.5.2 Biosynthesis and Accumulation of Chlorophyll b

Plastids were prepared from etiolated cucumber cotyledons at three different stages
of greening. In the first set of experiments, etioplasts were prepared from etiolated
cotyledons that were potentiated for Chl(ide) b biosynthesis by pretreatment with
one 2.5 ms flash of “actinic white light” followed by 60 min of dark incubation.
Such plastids were incapable, of Chl(ide) b net synthesis in vitro (Table 4.4 , A).
In a second set of experiments, the cotyledons were greened for 24 h
(120 nmol/m 2 /s of metal halide radiation) prior to the preparation of etiochloroplasts.
At this stage the cotyledons had accumulated large amounts of Chl a and b. The
evaluation of the Chl b biosynthetic activity in organello was rather uncertain because
of the high background of accumulated Chl a and b (Table 4.4 , B ) . In a third set of
experiments, etiolated cotyledons were greened for 4 h prior to etiochloroplast
preparation. At this stage of greening, the lag-phase of Chl b biosynthesis had been
removed and the tissue had just started active Chl b biosynthesis (Rebeiz 1967 ).
As a consequence, although Chl b biosynthesis was fully potentiated, the amount of
accumulated Chl(ide) a and b was not

large enough to interfere with the