Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 158

pH-Dependence of [4-Vinyl] Chlorophyllide a Reductase
Using Exogenous a DV Chlide a Substrate
Cucumber etioplast membranes were incubated at 30 C in various buffers covering
a pH range of 5.3-11.0. Mc Ilvaine buffer (40 mM citrate monohydrate/80 mM
K 2 HPO 4 ) was used for pH values ranging from 5.3 to 6.9. Tris-HCl (146 mM) was
used in the pH range of 7.0-9.0, and 0.25 mMK2HPO4/NaOH was used at pH 11.0.
All incubation buffers contained 0.2 %BSA and 0.55 mMNADPH. 4VCR exhibited
high activity between pH 6.0 and 7.0. No activity was observed at pH 11.0. After
2 min incubation at pH 11.0, no substrate degradation was observed at a pH of 6.3
and a temperature of 30 C, enzymatic activity in cucumber etioplast membranes
was quasilinear for the first 60 s of incubation (Parham and Rebeiz 1995 ).
[4-vinyl] Chlorophyllide a Reductase Activity is Expressed
in Other Plant Species
The occurrence of 4VCR in other plant species such as corn and barley was also
investigated. Unlike cucumber, which is a dark DV/light DV plant species (see
Chap. 14 ), corn and barley are two dark MV/light DV plant species (Ioannides
et al. 1994 ). In darkness, these monocotyledonous species accumulate MV Pchlide
a, and in the light they form MV Chl a . 4VCR activity was strongly expressed in
etiolated corn and barley. Contrary to the immediate onset of activity observed in
cucumber, an induction period of about 15 s was observed in barley and corn
etioplasts. The final levels of activity in corn and barley were higher than in
cucumber (Parham and Rebeiz 1995 ).
4.6.5.4 Solubilization and Partial Purification of 4-vinyl
Chlorophyllide a Reductase
Preparation of Etioplast Membranes
Percoll-purified etioplasts were suspended in lysing buffer at a rate of 30 ml per
pellet from 60 g of tissue. The lysing buffer consisted of 20 mM Tris-HCl, 1 mM
EDTA, 20 mM sucrose and 4 mM dithiothreitol, at a room temperature pH of
7.7. Etioplasts membranes were pelleted by centrifugation at 39,000 g for 12 min
at 2EC. One hundred g of etiolated barley leaves yielded 3.5-4.5 mg of membrane
protein.
Solubilization of 4VCR
Etioplast membranes were re-suspended in solubilization buffer at a rate of 2-3 mg
protein per ml. The solubilization buffer consisted of 20 mM Tris-HCl, 1 mM