Biology Reference
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cylindrical microcell 3 mm in diameter. Emission and excitation bandwidths of 4 nm
were used. The photon count was integrated for 0.5 s at each 1 nm increment.
The fluorescence excitation amplitude was converted to concentration by reference
to a calibration curve of known amounts of DV Chlide
a
versus fluorescence
excitation amplitudes. In constructing the calibration curve, the amount of DV
Chlide
a
was determined by room temperature absorption spectroscopy in 80 %
acetone, using a molar extinction coefficient of 69.29
10
3
at 663 nm (Shedbalkar
and Rebeiz
1992
). The proportion of DV and MV Chlide
a
was determined by 77 K
fluorescence excitation spectroscopy after transfer to diethyl ether as described
were recorded at emission bandwidths that varied from 0.5 to 4 nm depending on
signal intensity.
Conversion of Exogenous DV Chlide
a
to MV Chlide
a
by Membrane-Bound
[4-vinyl] Chlorophyllide
a
Reductase
Membrane-bound 4VCR was very active towards exogenous DV Chlide
a.
In eight
experiments, reaction rates ranged from 18.15 to 195.21 nmoles of MV Chlide
a
formed per 100 mg membrane protein per min. This range reflects biological
variations between various plastid membrane preparations. In each experiment the
samples were run in duplicate, and the difference between values for the two
samples averaged 5.70
5.32 nmoles per 100 mg membrane protein per min.
For the eight reported experiments, the mean values of substrate (DV Chlide
a
)
disappearance and product (MV Chlide
a
) formation were 74.11
9.14 and
62.80
4.04 nmoles per 100 mg membrane protein per min respectively. Product
formation from exogenous substrate was accompanied by about 15 % substrate
destruction. These rates are about 50-300-fold higher than the reported rates of
[4-vinyl] Pchlide
a
reductase towards exogenous Pchlide
a
in barley etioplasts
(Parham and Rebeiz
1995
; Tripathy and Rebeiz
1988
).
Temperature-Dependence of [4-Vinyl] Chlorophyllide
a
Reductase
Using Exogenous DV Chlide
a
Temperature-dependence of the enzyme was determined by incubating cucumber
etioplast membranes at 0, 20, 30, 40, and 60
C in 146 mM Tris-HCl buffer (pH 7.7)
containing 0.2 % BSA, and 0.55 mM NADPH. After temperature equilibration for
5 min, 4VCR activity was initiated by addition of DV Chlide
a
, to a final concentra-
tion of 1
M
.
Enzyme activity was monitored over a period of 2 min. Under these
conditions, maximal activity was observed at 30
C. Complete inhibition was
observed at 60
C. After 2 min incubation at 60
C, no substrate degradation
was observed.
μ
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