Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 161

pH 7.7 at room temperature. The resulting homogenate was filtered through four
layers of cheesecloth and centrifuged at 200 g for 5 min at 1 C in a Beckman JA-20
angle rotor. The supernatant containing etioplasts was decanted and centrifuged at
1,500 g for 10 min at 1 C. The pelleted etioplasts were gently resuspended with a
paintbrush in 2-ml of incubation buffer. The latter consisted of 500 mM sucrose,
20 mM MgCl 2 , 2.5 mM EDTA, 20 mM ATP, 40 mM NAD, 1.25 mM methanol,
200 mM Tris. HCl, 8 mM methionine, and 0.1 % BSA (w/v) adjusted with HCl to
a pH of 7.7 at room temperature. One ml of etioplast suspension was placed in a
25-ml beaker prior to light treatment.
The Petri dish containing excised, etiolated cucumber cotyledons, or the beaker
containing isolated etioplasts, were placed 10 cm below two 135 W second Rokunar
AC Studio Flash-Model 150 that generate a synchronized 2.5 ms actinic white
light flash. Reflecting mirrors were placed below and on the sides of the sample in
order to increase the amount of incident light reflected back onto the sample area.
Immediately after the flash, i.e. within less than 1 s, the treated tissues were frozen in
liquid N 2 (control), or left in darkness for different periods of time. At each dark-
time interval the reaction was stopped rapidly by pouring liquid nitrogen over the
tissue. For isolated etioplasts, the reaction was stopped by addition of 10 ml of
acetone:0.1 NNH 4 OH. Controls consisted of etiolated tissues or etioplasts that were
not subjected to the light treatment.
Effect of Exogenous Mg2 + on the Conversion of Chlorophyllide a
to Chlorophyll a
Mg 2+ is a cofactor which is involved in the conversion of: ALA to Pchlide a (Rebeiz
and Castelfranco 1971a ) of Proto to Mg-Proto, (Fuesler et al. 1981 ) and is usually
an adjunct cofactor in reactions involving ATP. It was therefore deemed desirable
to probe the effect of various concentrations of added MgCl 2 on the tetrapyrrole
biosynthetic capabilities of kinetin and GA-pretreated etiochloroplasts and on
the conversion of Chlide a to Chl a.
Pretreated etiochloroplasts responded very favorably to the addition of exogenous
MgCl 2 to the incubation medium. The rates of Pchlide and Proto net synthesis and
accumulation reached a maximum and a minimum, respectively, at an added Mg 2+
concentration of 20 mM while the highest rate of Mpe accumulation was observed
at an exogenous concentration of 40 mM. The requirement of different Mg 2+
concentrations for achieving optimal rates of Pchlide andMpe accumulation indicated
that in addition to insertion into Proto, Mg may also involved as a cofactor in
additional biosynthetic reactions between Mpe and Pchlide, most probably in the
formation of the cyclopentanone ring of Pchlide.
To our surprise, the conversion of Chlide a to Chl a was also found to dependent
on the addition of exogenous Mg 2+ (Daniell and Rebeiz 1984 ). This novel observa-
tion in turn indicated that in addition to ATP, Mg 2+ was also involved in the
conversion of Chlide a to Chl a. However, even at the highest rates of Chlide
a esterification, i.e. at a 20 mM concentration of added Mg 2+ , the conversion of