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Table 4.5 Detection of 4VMg-ProtoR and other 4VR activities in isolated plastid membranes and
solubilized fractions prepared from etiolated barley leaves
Net change in
MV Mg-proto
in 60 min
MV Mpe in
60 min
MV Pchlide
a in 60 min
MV Chlide a in
5 min
Membrane fraction
(pmoles per mg protein)
Membranes
29.2
14.1
48.2
19.7
243.8
16.8
47451
345.1
Solubilized fraction
155.9
30.6
235.9
30.6 1375.1
132.6 92211
345.1
Solubilized fraction as %
of Membrane activity
533.9
489.4
564.0
194.33
4VCR/4VR activities 591 391 67 1
Percoll-purified etiochloroplasts were isolated from barley seedlings under laboratory light.
The membranes and Chaps-solubilized fractions were prepared as described above
Values are means of 2-3 replicates
standard deviation
The activity of 4VMg-ProtoR was 591 time less than that of 4VCR
Percoll-isolated plastids. In this effort barley etiochloroplast membranes were used.
Barley etioplast membranes were prepared from crude and Percoll purified
etioplasts as described elsewhere (Kolossov and Rebeiz 2001 ). Solubilization of
4VMg-ProtoR activity was performed as described below.
Four millimolar Chaps was used for solubilization. Such a concentration
was successfully used in the past to solubilize 4-vinyl chlorophyllide reductase
(4VCR) (Kolossov and Rebeiz 2001 ). Essentially, membranes from Percoll
purified barley etioplasts were resuspended in solubilization buffer at a rate of 2 mg
protein per ml. The solubilization buffer consisted of 20 mM Tris-HCl, 1 mM
EDTA, 10 % of Glycerol and 4 mM 3-[(3-Cholamidopropyl)dimethylammonio]-1-
Propanesulfonate (Chaps) adjusted to a pH of 7.7 at room temperature. The remaining
steps were performed as described elsewhere (Kolossov and Rebeiz 2001 ).
Initial attempts at detecting 4-vinyl Mg-Proto reductase (4VMg-ProtoR or
4VMPR) in isolated etiochloroplast membranes under 4-vinyl Chlide reductase
(4VCR) incubation conditions (Kolossov and Rebeiz 2001 ) were unsuccessful.
After further experimentation, and adjustment of incubation conditions, it became
possible to detect 4VMg-ProtoR activity in the isolated membrane preparation.
However, the following adjustments in incubation conditions had to be made (a) the
incubation time for 4VMg-ProtoR was raised from 5 min to 60 min, (b) the sample
load was raised about tenfold; and, (c) the exogenous substrate concentration was
lowered 4-8 times. For 4VMPR however, the buffer was diluted 1:1 (v/v) with
distilled water in order to facilitate the extraction of Mg-Proto into diethyl ether.
The other incubation conditions were kept unchanged as described in (Kolossov
and Rebeiz 2001 ). The conversion of DV Mg-Proto to MV Mg-Proto by plastid
membranes and the solubilized 4VMg-ProtoR activity is reported in column 2 of
Table 4.5 which is displayed above.
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