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Other chemicals were tested for their effect on the biosynthetic activity of the
crude homogenate namely: ATP, NAD, NADP, thiamine pyrophosphate, cytidine
triphosphate, FAD, pyridoxal phosphate, NADPH, NADH, L-ascorbic acid,
dehydroascrobic acid, D,L-methionine, cytochrome c, D-glucose + glucose
oxidase, Fe 3+ ,Fe 2+, Zn 2+ ,Co 2+ , vitamin B 12 and mannitol. None of these produced
any stimulation in the cell-free crude homogenate system; some were slightly
inhibitory.
4.2.4.1 Effect of GSH and CoA on the Biosynthetic
Activity of the Crude Homogenate
The individual effects of added GSH and CoA on the biosynthetic activity of the
crude homogenate are described below. The omission of exogenous GSH from
the reaction mixture depressed the 14 C-incorporations into 14 C-Mpe, 14 C-Pchlide,
and 14 C-Pchlide ester regardless of the presence or absence of CoA. This finding
suggested a general protective effect of GSH on sulfhydryl enzymes and
porphyrinogen intermediates. However, the omission of exogenous CoA from the
reaction mixture containing GSH resulted in decreased 14 C-Pchlide biosynthesis
without interference with 14 C-Pchlide ester formation. It was conjectured that the
CoA site of action along the biosynthetic pathway was probably located after
Mpe formation and may be involved in the formation of 14 C-Pchlide but not of
I4 C-Pchlide ester. These results supported the hypothesis that Pchlide and Pchlide
ester were produced from a common precursor by two parallel and distinct
biosynthetic routes (Rebeiz et al. 1970 ).
4.2.4.2 Effect of K + and Pi on the Biosynthetic
Activity of the Crude Homogenate
In order to determine which component of the potassium phosphate buffer had
an effect on the biosynthetic activity of the system, the potassium phosphate
buffer was replaced by a tris-Pi buffer and K + was added back to the reaction
mixture as KCl.
Omission of exogenous phosphate had a depressing effect on the biosynthesis of
both 14 C-Mpe, 14 C-Pchlide, and 14 C-Pchlide ester (Rebeiz and Castelfranco
1971a ). On the other hand, the omission of exogenous potassium from the complete
mixture did not appear to affect the biosynthesis of 14 C-Pchlide ester but depressed
the biosynthesis of 14 C-Mpe and 14 C-Pchlide. The endogenous K + level in these
crude homogenates was, unknown. Although these results did suggest an involve-
ment of K + in the system, it was conjectured that additional experimental work with
washed etioplasts preparations was needed before a specific cofactor role could
be assigned to K + in Pchl(ide) biogenesis. The concentration of Cl was without
appreciable effect on the 14 C-ALA incorporation into Mpe, Pchlide, or Pchlide ester
(Rebeiz and Castelfranco 1971a ).
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