Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 150

Finally an attempt was made to determine whether the activity of Mg-Proto
chelatase, with the Proto substrate already adsorbed to the plastid membranes,
would increase upon addition of further exogenous Proto to the incubation medium.
No significant differences in Mg-chelatase activities were observed with the addi-
tion of various amounts of Proto to the incubation medium. This indicated that
the concentration of the Proto adsorbed to the membranes was high enough, to
saturate the chelatase activity during 2 h of incubation.
4.6.1.7 ATP Requirement for Subplastidic Membrane-Bound
Mg-Proto Chelatase Activity
In intact cucumber etiochloroplasts, Mg-Proto chelatase activity, became saturated
at about 10 mM ATP (Fuesler et al. 1984a ). Two sets of experiments were designed
to determine whether similar ATP concentrations would be required for optimal
activity of the subplastidic membrane-bound chelatase.
In one set of experiments, ATP was omitted from the lysing medium, while in
the other ATP was included in the lysing medium. When ATP was omitted from the
lysing medium prior to separating plastid membranes from stroma, Mg-Proto
chelatase activity was lost, irrespective of the amount of ATP subsequently added
to the incubation medium (Lee et al. 1992 ). This indicated that ATP was required
for enzyme stabilization during lysis and ultracentrifugation. When ATP was
included in the lysing medium, membrane-bound Mg-Proto chelatase responded
to addition of further ATP to the incubation medium in a manner similar to whole
etiochloroplasts. It exhibited optimum activity at about 15 mM ATP.
4.6.1.8 Mg requirement for Subplastidic Membrane-Bound
Mg-Proto Chelatase Activity
As reported for intact etiochloroplasts (Fuesler et al. 1984a ), added Mg ++ was also
required for Mg-Proto chelatase activity. Washing with EDTA prior to demon-
strating Mg requirement, as was reported by others for intact plastids (Fuesler
et al. 1984a ), was not necessary. After an initial lag phase that was overcome at
concentrations of MgCl 2 larger than 5 mM, the activity of Mg-Proto reached a
maximum at 10 mM MgCl 2 . Higher MgCl 2 concentrations were inhibitory
(Lee et al. 1992 ).
4.6.1.9 EDTA Requirement for Subplastidic Membrane-Bound
Mg-Proto Chelatase Activity
It has been our experience that optimal conversion of ALA to Proto, Mg-Porphyrins
and protochlorophyllide requires the presence of EDTA in both the homogenization
medium of greening tissues and the incubation medium of isolated etiochloroplasts