Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 149

(Lee et al. 1992 ). In other words, although Mg-Proto chelatase activity survived
etiochloroplast disruption, most of the activity was lost upon separating the plastid
membranes from stroma by ultracentrifugation. Several attempts were made to
stabilize Mg-Proto chelatase activity during ultracentrifugation. Success was
achieved when Proto, the natural substrate for Mg-Proto chelatase, was added to
lysed plastids immediately after lysis and prior to ultracentrifugation, at a concen-
tration of 100 nmoles/0.33 ml of lysed plastid suspension (Lee et al. 1992 ). It is very
likely that protection of Mg-Proto chelatase activity by adsorbed Proto involved
stabilization of the enzyme by its substrate, a well documented phenomenon
(Scopes 1982 ).
After ultracentrifugation all Mg-Proto chelatase activity was found in the mem-
brane fraction. The stroma was inactive. The isolated plastid membranes contained
the bulk of the added Proto. Although the observed membrane-bound Mg-Proto
chelatase activity (85.20 nmol/2 h/100 mg protein) was only one-sixth that of
purified etiochloroplasts, it was 145-450-fold higher than activities reported by
others for cucumber subplastidic preparations (Smith and Rebeiz 1977a ). It is worth
noting that no improvement in Mg-Proto chelatase activity was observed upon
recombining stroma and plastid membranes. On the contrary, the recombination
resulted in a statistically significant drop in activity.
4.6.1.6 Partition of the Exogenous Proto Substrate Between
the Membrane and Stromal Fractions
As reported above, exogenous Proto had to be added to the lysed etiochloroplasts to
stabilize the Mg-Proto chelatase activity during separation of plastid stroma from
membranes. After ultracentrifugation, about 80 % of the added Proto was found to
be associated with the membrane fraction, whereas the remaining 20 % was
recovered with the stroma (Lee et al. 1992 ). To determine whether the adsorbed
Proto was loosely or tightly bound to the membranes, the latter were resuspended in
the lysing medium and were subjected to a second ultracentrifugation. Almost all
the adsorbed Proto was recovered in the membrane fraction thus indicating that the
Proto was tightly associated with the plastid membranes.
Further insight into the molecular environment of the membrane-bound Proto
was derived from fluorescence polarization and anisotropy measurements. It is
acknowledged that slow rotation of a fluorophore such as Proto, relative to the
rapid emission of fluorescence, results in larger polarization and anisotropy values
than if the fluorophore is rapidly undergoing rotation. On the other hand, the rate of
rotation of a fluorophore depends on its molecular environment. For example a
fluorophore in a viscous or rigid environment rotates much slower than in a more
fluid environment. The polarization and anisotropy values of membrane-bound
Proto were significantly higher than for the stromal Proto or for Proto dissolved
in 80 % aqueous acetone or in the aqueous incubation medium (Lee et al. 1992 ).
This suggested that the membrane-bound Proto was most probably solvated in a
more rigid environment, such as the hydrophobic core of the membrane fraction.