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Table 4.1 Optimum concentrations of various cofactors needed for the Biosynthesis of Pchlide,
Mpe and Proto by isolated plastids
Optimum cofactor concentration (mM) for:
Pchlide
Cofactors
MP(E)
Proto
Adenosine 5
0
-triphosphate
20.0
20.0
0
Nicotinamide adenine dinucleotide (oxidized)
40.0
0
0
Glutathione, reduced
0
10.0
5.0
Coenzyme A
0
0
0
Methyl alcohol
1.25
2.5
25.0
EDTA
2.5
1.25
10
MgCl
2
20.0
20.0
20.0
KH
2
PO
4
0
0
0
1 %
a
Bovine serum albumin
5%
0
Sucrose
330.0
330.0
330.0
a
1 % (weight/volume)
Adapted from Rebeiz et al. (
1982
)
The tissue was hand-homogenized (ten strokes) at 4
C and the plastids were
incubated in the dark for 2 h in the absence or presence of different concentrations
of various cofactors. Each incubation consisted of 2 ml of plastids (4-6 mg protein),
0.1 ml of 10 mM ALA, and 0.9 ml of H
2
O. The Bovine serum albumin amounted to
a
1 % (weight/volume). Table
4.1
, is displayed above.
4.4.2 Effect of Kinetin in Enhancing the Synthesis
and Accumulation of Protochlorophyllide in Organello
We had previously proposed that etiochloroplasts differentiated less satisfactorily
in organello than in vivo partly because Pchl(ide) and prothylakoid membranes
accumulation appeared to be limited in vitro, by structural proteins synthesized in
the cytoplasm and in the absence of which the massive formation of prothylakoids
and grana was not possible (Rebeiz et al.
1973
). It was therefore conjectured that
should a method be found for obtaining etiochloroplasts containing excess Pchl
(ide)-binding prothylakoids but lacking stochiometric amounts of membrane-bound
Pchl(ide) then these plastids may be able to synthesize Pchlide, in organello, at very
high rates in order to saturate the Pchl(ide) binding sites. We had indeed
demonstrated earlier that in etiochloroplasts all of the Pchl(ide) was membrane
bound (Smith and Rebeiz
1979
). Several independent observations suggested that
the forementioned goal may be experimentally feasible. First we noticed that when
excised etiolated cucumber cotyledons were incubated overnight in the dark with an
aqueous solution of kinetin, they underwent a 370 % increase in size. However,
their Pchlide content increased only by about 128 % On the other hand, cytokinins
are known to (a) promote the differentiation of plastids in vivo (Stetler and Laetsch
1965
), and (b) as mentioned in (Daniell and Rebeiz
1982a
) to increase the size and
number of chloroplasts per cell and to increase the rate of RNA DNA and protein
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