Biomedical Engineering Reference
In-Depth Information
9.5.3 Cell Membrane Components
While testing different cells for the capacity to regenerate secreted CT transformation
activity (Table
9.1
), Tatara (
1996
) discovered one cell type,
Lacobacillus acidophilus
, that was
unable to regenerate the secreted PDTC.
L. acidophilus
is a strictly fermenting organism, and
lacks electron transport chains, suggesting a possible role of electron transport chains in
activation of the secreted PDTC for CT transformation (later identified as the Cu:PDTC
complex). To investigate this possibility, Tatara prepared crude cell membrane and cytoplasmic
fractions from strain KC cells, then combined them with the secreted PDTC to determine
whether membrane bound or cytoplasmic proteins enhance CT transformation. Reduced
nicotinamide adenine dinucleotide (NADH) also was added to determine whether it enhances
transformation and, if so, whether it does so in combination with an additional mediator
protein. Freshly prepared membrane and cytoplasmic fractions were combined with purified
supernatant with and without NADH. As shown in Figure
9.5
, rapid CT transformation
occurred when secreted PDTC (labeled here as SF for secreted factor) was combined with
crude cell membranes in the presence of 20
m
M NADH. Transformation of CT also occurred
when secreted PDTC was combined with crude cell membranes without NADH, but rates were
not as fast as with NADH. No CT transformation was observed when secreted PDTC was
combined with the cytoplasm of stain KC cells with or without NADH present. The secreted
PDTC did not mediate significant CT transformation when combined with NADH, indicating
that rapid transformation required a membrane-associated protein and transfer of reducing
equivalents from NADH.
Tatara (
1996
) also tested the effects of inhibitors of respiration, including antimycin
and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), and the protonophore, carbonyl cyanide
m
-chlorophenyl hydrazone (CCCP). HQNO inhibits respiration by blocking electron transport
between coenzyme Q and cytochrome b, and antimycin inhibits electron transport between
cytochrome b and cytochrome c or cytochrome o. No inhibition of CT transformation was
found in the presence of antimycin, HQNO or CCCP. This result indicates that coupling via a
proton gradient does not directly drive CT transformation, and implies that any electron
0.20
SF + KC
cytoplasm
SF + KC cyto. +
20 µM NADH
0.15
SF + crude KC
membranes
0.10
SF + P.fluor.
cells
SF + crude KC
membranes
+ 20
µ
M NADH
0.05
0.00
0
30
60
90
120
150
Time (min)
Figure 9.5. Transformation of CT by PDTC secreted by strain KC (SF) in the presence of crude cell
membrane and cytoplasm lysate. The ß-form of NADH was used in this experiment. Error bars
represent the standard deviation of triplicate sample.
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