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19.3.2 Protoporphyrinogen Accumulation
in the Mitochondria of MLA 144 Cells
Treated with ALA and Oph
Homogenates of MLA 144 cells treated with 1 mM ALA and 0.75 mM Oph for 3 h
in darkness accumulated significant amounts of protoporphyri(nogen) [Proto(gen)]
i.e. a mixture of Proto and its reduced hexahydro analog protoporphyrinogen
(Protogen), but did not accumulate other porphyrins. After oxidation of Protogen
to Proto, the latter was identified by its fluorescence emission and Soret absorption
maxima at 632 nm and 404 nm, respectively, in hexane-extracted acetone at room
temperature (Rebeiz et al. 1992 ). Proto was also identified by chromatography
with standard Proto on a Waters Bondapak reverse-phase C18-bonded column
(Rebeiz et al. 1992 ).
Following subcellular fractionation of the treated cells, the Proto(gen) content
of each subcellular fraction was determined. The total Proto(gen) content of
treated cells averaged 221 18 nmol/100 mg protein (n ¼ 6). The 1,000 g
pellet, which contains mainly nuclei, plasma membranes and unbroken cells
accumulated significantly less Proto(gen) (95
10 nmol/100 mg protein). The
bulk of
the biosynthesized Proto(gen) accumulated in the mitochondria
(235
45 nmol/100 mg protein, p
0.05). Some Proto(gen) accumulated in
<
the ER/microsomes (62
8.7nmol/100mgprotein),but very little was observed
in the cytoplasm (9
2 nmol/100 mg protein) (Rebeiz et al. 1992 ). These results
confirmed that in MLA 144 cells mitochondria were the primary site of Proto
biosynthesis (Dailey 1990 ). It was conjectured that the Proto found in the
ER/microsomes may be due to contamination by broken mitochondria and/or to
transport of Proto(gen).
19.3.3 Biosynthetic Origin of Protoporphyrinogen
Accumulation in the Mitochondria
To determine the biosynthetic origin of Proto(gen) accumulation in the
mitochondria, the ability of isolated MLA 144 subcellular fractions to synthesize
porphyrin(ogen)s from ALA in the presence of Oph was investigated.
In addition to Proto(gen), the formation of other porphyrin(ogen)s was
observed. Prior to identification, porphyrinogens were converted to porphyrins as
described in (Rebeiz 2002 ). The oxidized form of coproporphyrinogen (Coprogen),
i.e. copropporphyrin (Copro), was identified by its fluorescence emission and Soret
excitation at 620 and 394 nm, respectively, in hexane-extracted acetone at room
temperature (Rebeiz et al. 1975 ) and by chromatography with standard Copro on a
Waters Bondapak reverse-phase C18-bonded column (Rebeiz et al. 1996a ). Other
oxidized porphyrinogens such as uroporphyin III (Uro) (RT
3.4 min) as well as
¼
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