Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 147

for 20 min at 1 C. The pelleted crude etiochloroplasts were gently resuspended in
5 ml of homogenization, suspension or lysing medium using a small paintbrush.
The suspension medium was composed of 500 mM sucrose, 200 mM Tris, 20 mM
MgCl2, 2.5 mM EDTA, 40 mM NAD + , 20 mM ATP, 8 mM methionine, 1.25 mM
methanol, and 0.1 % (w/v) BSA at a room-temperature pH of 7.7. Unless otherwise
indicated, the lysing medium consisted of 25 mM Tris, 30 mM MgCl2, 7.5 mM
EDTA, 40 mM NAD+, 20 mM ATP, 8 mM methionine, 37.5 mM methanol, and
4.5 mM glutathione at a room-temperature pH of 7.7 (Lee et al. 1992 ). For further
plastid purification, the pelleted crude etiochloroplasts were resuspended in 5 ml
of homogenization medium and were purified by Percoll density centrifugation
(Lee et al. 1992 ). The pelleted, Percoll-purified etiochloroplasts were then
resuspended either in the suspension or lysing medium.

4.6.1.2 Preparation of Etiochloroplast Stroma and Membranes

To stabilize Mg-Proto chelatase activity, and unless otherwise indicated,
100 nmoles of Proto per 0.33 ml of membrane suspension were added to the
lysed plastids immediately after lysis. The stroma and membranes fractions were
then resolved following ultracentrifugation at 235,000 g for 1 h in a Beckman 80 Ti
angle rotor at 1 C (Lee et al. 1992 ).

4.6.1.3 Mg-Proto Chelatase Assay

In a total volume of 1 ml containing 0.33 ml of crude etiochloroplasts, or purified
etiochloroplasts,

lysed plastids, stroma, or membrane fractions, the reaction

mixture consisted of 100 M Proto, 330 mM sucrose, 200 mM Tris, 20 mM

MgCl2, 5 mM EDTA, 27 mM NAD + , 15 mM ATP, 5 mM methionine, 25 mM
methanol, 3 mM glutathione, and 0.1 % (w/v) BSA, at a room-temperature pH of
7.7. Incubation was in a flat-bottom glass tube. To each incubation tube was added
0.01 ml of 10 mM Proto (100 nmoles) except when the Proto had already been
added to the lysed etiochloroplast suspension. The tubes were wrapped in alumi-
num foil and were incubated at 28 C for 2 h in darkness in a shaking water bath
operated at 50 oscillations/min.
Before and after incubation, pigments were extracted by the addition of 5 ml of
cold acetone:0.1 N NH4OH (9:1 v/v) per ml reaction mixture. This was followed by
centrifugation at 39,000 g for 10 min at 1 C. The ammoniacal acetone extract
was retained, and the pellet was discarded. 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 containing Proto,
Mg-Proto, and Pchlide, was used for quantitative determination of Mg-Proto by
spectrofluorometry (Rebeiz et al. 1975b ). The measured Mg-Proto pool consisted of
Mg-Proto and smaller amounts of Mg-Proto monoester.

μ