Biology Reference
In-Depth Information
heptaporphyrins (RT
4.2 min) and hexaporphyrins (RT
5.8 min) were formed
¼
¼
during the conversion of Urogen to Coprogen.
Incubation of MLA 144 whole cell homogenates with ALA and Oph in darkness,
resulted in the formation of Proto and lesser amounts of Uro(gen), heptaporphyrin
(ogen) and Copro(gen) (Rebeiz et al.
1996a
). However, incubation of the cytoplas-
mic fraction with ALA + Oph resulted mainly in the biosynthesis and accumulation
of Uro(gen), heptaporphyrin(ogen) and Copro(gen), and lesser amounts of Proto
(gen) and hexaporphyrin(ogen).
On the other hands Isolated mitochondria treated with ALA (1 mM), Oph
(0.75 mM) and ATP (15 mM) formed very little Proto(gen) (Rebeiz et al.
1996a
).
Some Copro(gen) accumulation was observed (10 pmol/5 ml reaction) which might
be due to contamination of the mitochondrial fraction by low levels of cytoplasm/
ER. The cytoplasmic/ER fractions accumulated significant amounts of Copro(gen)
(25 pmol/5 ml reaction; p
0.05) and much smaller amounts of Proto(gen).
More significantly, when the cytoplasmic/ER and mitochondrial fractions were
combined, in addition to Copro(gen), significant amounts of Proto were formed
(Rebeiz et al.
1996a
).
Similar results as those described above were observed when cytoplasm alone,
and cytoplasm + mitochondria were incubated with ALA (1 mM), Oph (0.75 mM)
and ATP (15 mM), in the absence of added ER. Altogether, these results indicated
that Proto biosynthesis and accumulation from ALA in MLA 144 cells required the
cooperation of mitochondria and cytoplasm.
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19.3.4 Cofactor Requirement for the Biosynthesis
and Accumulation of Protogen by Mitochondria
To gain better understanding of the cooperation of mitochondria and cytoplasm
during the biosynthesis and accumulation of Proto(gen), in mitochondria, the
cofactor requirement of this process was investigated. Preliminary experiments
indicated that the conversion of ALA to Protogen by mitochondria + cytoplasm
was ATP-dependent (Rebeiz et al.
1996a
). Thus the possibility that Coprogen,
formed in the cytoplasm, may be actively transported into the mitochondria
where it is converted to Proto was therefore investigated. Coprogen (1,250 pmol/
ml) was incubated with isolated mitochondria in the presence or absence of ATP. In
the absence of ATP, some Proto biosynthesis was observed (29
11.5 nmol/
100 mg protein). However, at 100 mM of exogenous ATP, a 62 % increase in
Proto formation was observed in comparison to controls (p
0.05), thus indicating
that ATP enhances the conversion of exogenous Coprogen to Proto by
mitochondria. The high concentration of added ATP may have been mandated by
the high concentration of the added Coprogen substrate.
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