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Table 4.6 NADPH requirement of membrane-bound 4VCR
Net change in DV Chlide
a
in 20 min
Net change in MV Chlide
a
in 20 min
Etiochloroplast membranes
a
(nmol/100 mg protein)
b
2.1 (
19.8 %)
c
2.9
d
Without added reductants
with added NADPH
19.8 (
94.3 %)
20.5
With added NADH
2.9 (
24.6 %)
3.8
With added GSH
3.1(
25.8 %)
3.0
a
Etiochloroplasts were lysed in lysing buffer containing 25 mM Tris-HCl
b
Mean of two replicates
c
Values in parenthesis represent the net change in DV Chlide
a
, as a percent of total DVChlide
a
present before incubation
d
In all cases, either no MV Chlide
a
or small amounts of MV Chlide
a
were detected at the
beginning of dark incubation
plastid suspension was centrifuged at 235,000 g for 1 h in a Beckman 80 Ti fixed angle
rotor at 1
C. This centrifugation separated the suspension into a colorless soluble-
protein supernatant (stroma) and a yellowish pellet (membranes) (Lee et al.
1991
).
The stromal fraction was decanted and the pelleted membranes were either
resuspended in the lysing medium at a rate of 5 ml per 5 g of homogenized tissue or
were washed once before use. To this effect, plastid membranes from 5 g of tissue
(about 3 mg) were resuspended in 8 ml of 146 mM Tris-HCl adjusted to a room
temperature pH of 7.7, and containing all the additives present in the lysing buffer.
The suspension was centrifuged at 235,000 g for 0.5 h at 1
C. The supernatant
was decanted and the pelleted membranes were resuspended in 3 ml of lysing buffer
containing or lacking cofactors.
DV Chlide
a
reduction was initiated by conversion of most of the DV Chlide
a
to MV Chlide
a
by a single, 2.5-ms flash of actinic white light followed by a dark
incubation (Duggan and Rebeiz
1982a
). The reduction of DV Chlide
a
to MV
Chlide
a
was allowed to proceed in darkness for 20 min.
Conversion rates in the presence and absence of added NADPH are reported in
Table
4.6
, which is displayed above.
4.6.5.3 Conversion of exogenous DV Chlide a to MV Chlide
a in Isolated Cucumber Etiochloroplast Membranes
Preparation of Plastid Membranes
Plastids were isolated essentially as previously described in (Parham and Rebeiz
1992
). Essentially plastid isolation was performed under a safe green light that
transmitted light between 510 and 520 nm, and which did not photoconvert Pchlide
a
to Chlide
a
. Five g batches of etiolated cucumber cotyledons, and etiolated barley,
or corn leaves were hand homogenized in a cold mortar. The tissue was ground in
12.5 ml of homogenization buffer. The latter consisted of 500 mM sucrose, 15 mM
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