Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 153

4.6.3 Development of a Cell-Free System Capable
of the Conversion of Divinyl Mg-Proto Ester
to Monovinyl Mg-Proto Ester
This enzyme was first reported by Ellsworth and Hsing in a supernatant of etiolated
wheat leaves homogenates (Ellsworth and Hsing 1973 ), but was never confirmed by
others (Rebeiz et al. 2003 ).
In contrast to previous reports (Ellsworth and Hsing 1973 ), and for the first the
time, 4VMpeR activity was unambiguously demonstrated as a membrane-bound
enzyme. The activity was detected under the same preparative and incubation
conditions described above for 4VMg-ProtoR. The 4V-MpeR activity is reported
in column 3 of Table 4.5 . The activity was 391 times weaker than that of 4VCR.
4.6.4 Development of a Cell-Free System Capable
of the Conversion of Divinyl Protochlorophyllide
to Monovinyl Protochlorophyllide
4.6.4.1 Conversion of DV Pchlide a to MV Pchlide
a in barley etiochloroplasts
The dark conversion of exogenous DV Pchlide to MV Pchlide in barley plastids
poised in the DV monocarboxylic biosynthetic mode was investigated in barley
etiochloroplasts as well as in barley chloroplasts. Isolated etiochloroplasts prepared
from barley leaves preirradiated for 5 h were incubated in a medium that consisted
of 0.5 M sucrose, 0.2 M Tris-HCl, 20 mM MgCl2, 2.5 mM Na 2 EDTA, 20 mM
ATP, 40 mM NAD, 8 mM methionine, 0.1 % bovine serum albumin (w/w), and
1.25 mM methanol at a room temperature PH of 7.7 (Tripathy and Rebeiz 1988 ).
After 1 h of incubation. The etioplasts converted about 24-27 nmole per 100 mg
plastid protein of DV Pchlide to MV Pchlide. Isolated barley chloroplasts converted
1.5 nmoles (Tripathy and Rebeiz 1988 ).
4.6.4.2 Conversion of DV Pchlide a to MV Pchlide
a in Isolated Barley Etiochloroplast Membranes
The rates of conversion of DV to MV Pchlide increased significantly in solubilized
4V-PideR prepared as described above in for 4V-Mg-Proto and its ester. The
high rates of conversion are described in column 4 of Table 4.5 (Kolossov and
Rebeiz 2010 ). The whole activity was confined to the inner barley etiochloroplast
membranes, none was detected in the etiochloroplast envelope (Kolossov and
Rebeiz 2010 ). Furthermore the activity was completely dependent on the presence
of NADPH. The activity was also detected in barley chloroplasts (Kolossov and
Rebeiz 2010 ).