Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 148

Fluorescence spectra were recorded at room temperature on a fully corrected
photon-counting SLM spectrofluorometer Model 8000C, interfaced with an IBM
model XTmicrocomputer. Determinations of Mg-Proto were performed on an aliquot
of the hexane-extracted acetone fraction in a cylindrical microcell 3 mm in diameter.
All spectra were recorded at emission and excitation bandwidth of 4 mm. The amount
of Mg-proto was determined from its fluorescence amplitude at its emission maxi-
mum, upon excitation at 420 nm. Fluorescence amplitudes were converted to
Mg-Proto concentrations by reference to a standard calibration curve. The digital
spectral data were automatically converted by the computer into quantitative values.
Fluorescence polarization and anisotropy of Proto in different environments
were measured by simultaneously observing the horizontal and vertical emission
from the sample when exciting with horizontally and vertically polarized light as
described in the SLM manual.
4.6.1.4 Demonstration of Mg-Proto Chelatase Activity
in Ruptured Etiochloroplasts
The purity of Percoll-purified cucumber etiochloroplasts and the efficacy of lysis by
osmotic shock were evaluated in (Lee et al. 1991 ). Percoll-purified etiochloroplasts
were more highly intact (87 %) than crude etiochloroplasts (68 %) and contamina-
tion by other subcellular organelles was reduced five to ninefold in comparison to
the crude etiochloroplasts. Lysis of etiochloroplasts by osmotic shock was as
efficient (98 %) as lysis by 0.1 % Triton X-100 (100 %) (Lee et al. 1991 ).
The activities of crude and Percoll-purified etiochloroplasts amounted to 325 and
540 nmoles respectively of Mg-Proto synthesized per 100 mg of plastid protein.
These values were two to threefold larger than those reported by others for develop-
ing cucumber chloroplasts (Fuesler et al. 1981 , 1984a ; Walker and Weinstein
1991a ). No significant differences in Mg-Proto chelatase activity between unlysed
and lysed etiochloroplasts were observed, although the activity of purified plastids
were significantly higher than the crude ones. It was therefore concluded that the
Mg-Proto chelatase activities of either crude or Percoll-purified etiochloroplasts
were not altered by plastid rupture. This in turn indicated that in cucumber
etiochloroplasts, plastid intactness was not a mandatory requirement for the inser-
tion of Mg 2+ into Proto by Mg-Proto chelatase. This was at variance with the results
of others who found that any disruption of cucumber chloroplasts resulted in a
drastic decrease in Mg-Proto chelatase activity (Fuesler et al. 1984b ; Walker and
Weinstein 1991a ).
4.6.1.5 Stabilization of Mg-Proto Chelatase Activity
in a Subplastidic Membrane Fraction
Initial attempts aimed at recovering Mg-Proto chelatase activity in isolated
subplastidic fractions met with limited success. Some activity was recovered
in unstabilized plastid membranes and none was found in the plastid stroma