Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 151

(Rebeiz et al. 1982 ). When EDTA was omitted from the homogenization, lysing,
and incubation media, Mg-Proto chelatase activity was lost and membrane-bound
Proto was converted to Zn-Proto (emission maximum at 590 nm, in hexane-
extracted acetone, at room temperature), instead of being converted to Mg-Proto
(emission maximum at 595 nm). The fluorescence properties of Mg-Proto and
Zn-Proto have been described elsewhere (Smith and Rebeiz 1977b ). Addition of
2.5-20 mM EDTA to the incubated plastids did not restore the Mg-Proto chelatase
activity, but suppressed Zn-Proto formation. When EDTA was included in the
homogenization, and lysing media, addition of EDTA (2.5-10 mM) to the incuba-
tion medium did not affect Mg-Proto chelatase activity, which appeared to proceed
normally. However higher concentrations of added EDTA (15-20 mM) severely
inhibited (74-83 %) Mg-Proto formation.
4.6.1.10 Lack of Effect of Other Additives on Mg-Proto
Chelatase Activity
The lysing medium also contained NAD + , gluthathione, methanol and methionine,
the effect of which upon Mg-Proto chelatase activity had not been determined.
Percoll-purified plastids were therefore lysed in the normal lysing medium which
contained NAD + , glutathione, methanol and methionine, or in a lysing medium
which lacked the above additives. The separated membranes were then incubated in
the presence and absence of the aforementioned additives. These additives
exhibited no measurable effect on Mg-Proto chelatase activity (Lee et al. 1992 ).
4.6.2 Development of a Cell-Free System Capable
of the Conversion of Divinyl Mg-Protoporphyrin
IX to Monovinyl Mg-Protoporphyrin IX
Kim et al. ( 1997 ) had reported earlier that an interaction of plastid membranes,
stroma, and NADPH was involved in the regulation of DV and MV Pchlide
a biosynthesis. Although the combination of plastid stroma and plastid membranes
resulted in higher total Pchlide a formation from exogenous DV Mg-Proto, the
presence of stroma in the reaction mixture resulted in a 5 to16-fold decrease in
the MV/DV Pchlide a ratio. This in turn suggested an inhibition of 4-vinyl reduc-
tase (4VR) reactions between DV Mg-Proto and Pchlide a by the plastid stroma.
It was therefore conjectured that the study of 4VR activities in the absence of the
plastid stroma, i.e. in isolated plastid membranes, may give a deeper insight into
4VR activities and may unmask some additional undetected 4VR activities.
It was therefore conjectured that a comparative study of various vinyl reductase
(4VR) activities would be facilitated by solubilization of 4VR activities from