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However resting and Con A-activated splenocytes, as well as MLA 144 cells,
treated with medium alone, did not accumulate any detectable amounts of Proto
(Rebeiz et al.
1992
).
19.3
Intracellular Localization of Heme Biosynthesis in
Animal Cells
Since Proto accumulation is the essential factor in causing photodynamic damage in
animal cells, the next three sections investigated its intracellular accumulation in
mammalian cells.
The intracellular site of heme biosynthesis in mammalian cells has been investigated
by (Granick
1967
). Using ox liver mitochondria, Granick proposed the following
scheme: ALA is formed in the mitochondria and then translocates out of the
mitochondria into the cytoplasm. In the cytoplasm, ALA is converted to copropor-
phyrinogen III (Coprogen III). Then Coprogen III translocates back into the mitochon-
drion where it is converted to Proto and heme. While this hypothesis has been popular
for many years, it is beset with problems. Translocations into and out of the mitochondria
are energetically wasteful. Secondly, since no evaluation of mitochondrial breakage
during mitochondria isolation has been provided, it was difficult to evaluate contamina-
tion of the cytoplasm by mitochondrial content. It was therefore decided to reevaluate
this hypothesis in neoplastic cells as described below.
19.3.1 Purity of the Mitochondrial Preparations
First the purity of the mitochondrial preparations used in the studies was evaluated.
Subcellular organelles were isolated from MLA 144 cells by differential centrifuga-
tion in sucrose buffer. To determine the purity of the mitochondrial fraction, the
activity of three marker enzymes for mitochondria, cytoplasm and endoplasmic
reticulum (ER)/microsomes was determined. Succinate cytochrome c reductase
(SCR), a mitochondrial marker located the inner mitochondrial membrane, was
used as an indicator of mitochondrial activity. Washed mitochondria exhibited a
significant enrichment in SCR activity in comparison to the cell homogenate (Rebeiz
et al.
1996a
). There was little or no SCR activity in the cytoplasm and ER/microsomes,
thus indicating that mitochondria were not present in these fractions (Rebeiz
et al.
1996a
). In addition, hydroxypyruvate reductase (HPR), an ER/microsomal
marker and lactate dehydrogenase, a cytoplasmic marker, were significantly lower
in the mitochondrial fraction (Rebeiz et al.
1996a
). These results indicated that it was
possible to prepare mitochondria from MLA 144 cells with reduced contamination
by cytoplasm and ER/microsomal fractions.
Next it was determined whether mitochondria were the site of Proto accumula-
tion as described below.
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