Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 135

4.2.4.5 Intracellular Localization of Mg Protoporphyrin

and Pro-tochlorophyll Biosynthesis

In order to establish a connection between the porphyrin and phorbin biosynthetic
activities and various subcellular fractions, the crude homogenate was fractionated
by differential centrifugation into a crude etioplast preparation and a supernatant,
enriched in soluble proteins, microsomes, mitochondria, and microbodies. The
crude etioplast preparation was more active than either the crude homogenate or
the supernatant (Rebeiz and Castelfranco 1971a ).
Also a crude homogenate was prepared from etiolated cotyledons in a grinding
buffer containing all the cofactors needed for maximal Pchl(ide) activity. From this
homogenate a fortified etioplast pellet was prepared which was much more active
than anything we had previously encountered and had a specific radioactivity
much higher than the fortified crude homogenate (Rebeiz and Castelfranco
1971a ). Upon washing these crude fortified etioplasts with the fortified grinding
buffer their biosynthetic activity was remarkably well preserved (Rebeiz and
Castelfranco 1971a ). These results indicated that the in vitro biosynthesis of
14 C-Mpe, 14 C-Pchlide, and 14C-Pchlide ester was associated with the etioplasts.

4.2.4.6 ATP and NAD Requirement for Maximal Biosynthetic
Activity of Washed Fortified Etioplasts

The ability to prepare washed, active etioplasts presented a good opportunity for
further studies of cofactor requirements in the presence of reduced levels of endoge-
nous cofactors. Although a limited amount of experimentation was performed on this
particular system, a requirement for ATP and NAD was established (Rebeiz and
Castelfranco 1971a ). It appeared that in the presence of ATP and NAD, the utilization
of 14 C-Mpe was increased as evidenced by reduced levels of the latter and increased
levels of 14 C-Pchlide. Both ATP and NAD appeared to be required for maximal
accumulation of l4 C-Pchlide by the washed fortified etioplasts (Rebeiz and
Castelfranco 1971a ). The presence of ATP did not appear to stimulate 14 C-Pchlide
ester accumulation. On the other hand, NAD alone, in the absence of ATP, resulted in
a marked increase of l4 C-Pchlide ester formation (Rebeiz and Castelfranco 1971a ).
These observations indicated that although both ATP and NAD were required for
14 C-Pchlide biosynthesis, only NAD was needed for 14 C-Pchlide ester biosynthesis.

4.3 Chlorophyll Biosynthesis in Organello

Once total 14 CPchl(ide) biosynthesis in crude homogenates and in organello was
achieved (Rebeiz and Castelfranco 1971a ). We undertook the total biosynthesis
of Chl in organello. Using the Pchl(ide) biosynthetic system it was possible to
achieve total Chl biosynthesis in organello by carrying the incubations in the light
instead of in darkness, since light is required for Chl biosynthesis.