Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 156

Hepes, 1 mM MgCl 2 , 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 in a Beckman JA-20 fixed angle rotor at 1 C.
The supernatant was decanted and centrifuged at 1,500 g for 10 min at 1 C. The
pelleted etiochloroplasts (about 3-5 mg protein) were osmotically lysed in 5 ml of
dilute (lysing) buffer consisting of 25 mM Tris-HCl, and 0.2 % BSA. The pH was
adjusted to 7.7 at room temperature. The resulting suspension was centrifuged at
235,000 g for 1.0 h in a Beckman 80 Ti fixed-angle rotor at 1 C. The pelleted
membrane fraction was separated from the stroma and resuspended with a paint-
brush in incubation buffer at a ratio of 5 ml/5 g of tissue. Unless otherwise indicated
the incubation buffer consisted of 40 mM citrate monohydrate:80 mM K 2 HPO 4 ,
0.2 % BSA, and 0.55-0.80 mM NADPH, at pH 6.3. For determination of the
optimum pH of the enzyme, other incubation buffers were used as indicated below.

Assay of [4-vinyl] Chlorophyllide a Reductase
Using Exogenous DV Chlide a

All steps were carried out under a green safelight that transmitted light between
510 and 520 nm, and which did not photoconvert Pchlide a to Chlide a . Etioplast
membranes devoid of any endogenous Chlide a or b were isolated and resuspended
in 1 ml of an NADPH-fortified incubation buffer as described above. To achieve
temperature equilibration, the reaction mixture was pre-incubated for 5 min at
30 C before initiating the reaction. The reaction was triggered by addition of
25-30 l of DV Chlide a substrate dissolved in 80 % acetone, to a final concentra-

tion of 1

μ

M, and was monitored spectrofluorometrically (see below) by the
appearance of MV Chlide a , the reaction product . Depending on the particular
experiment, the reaction was allowed to proceed for 45 s to several min in darkness,
and was terminated by addition of 7 ml of cold ammoniacal acetone. The resulting
mixture was centrifuged at 39,000 g for 12 min at 1 C. Chlorophylls and other fully
esterified tetrapyrroles were transferred from acetone to hexane by extraction with
an equal volume of hexane, followed by a second extraction with one-third volume
of hexane. The remaining hexane-extracted acetone residue, which contained
monocarboxylic and dicarboxylic tetrapyrroles, was used for pigment determina-
tion by spectrofluorometry at room temperature and at 77 K. Spectrofluorometric
determinations at 77 K were performed after transfer of the pigments to ether .
The amount of DV and MV Chlide a was determined from the total amount of
Chlide a and from the proportion of DV and MV Chlide a , as described below
(see Chap. 3 ) . The total amount of Chlide a was determined on an aliquot of the
hexane-extracted acetone fraction from the room temperature Soret excitation
maximum at 433 nm. The room temperature fluorescence excitation spectrum was
recorded at an emission wavelength of 674 nm on a fully corrected photon counting,
high-resolution SLM spectrofluorometer Model 8000C, interfaced with an IBM
microcomputer Model 60. The hexane-extracted acetone aliquot was placed in a

μ