PLANT PHYSIOLOGY , Vol 104, Issue 2 381-386, Copyright © 1994 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Antigenic Relationships between Chloroplast and Cytosolic Fructose-1,6-Bisphosphatases
J. Fonolla, R. Hermoso, J. L. Carrasco, A. Chueca, J. J. Lazaro, F. E. Prado and J. Lopez-Gorge
Department of Plant Biochemistry, Estacion Experimental del Zaidin, Consejo Superior de Investigaciones Cientificas, 18008-Granada, Spain
Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum
sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant)
did not cross-react by double immunodiffusion and western blotting with
either of the antisera raised against the chloroplast enzyme of both
species; similarly, pea and spinach chloroplast FBPases did not react with
the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea
chloroplast FBPases showed strong cross-reactions against the antisera to
chloroplast FBPases, in the same way that the pea and spinach cytosolic
enzymes displayed good cross-reactions against the antiserum to spinach
cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as
the corresponding purified chloroplast enzymes, showed by western blotting
only one band (44 and 43 kD, respectively) in reaction with either of the
antisera against the chloroplast enzymes. A unique fraction of molecular
mass 38 kD appeared when either of the crude extracts or the purified
spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase
antiserum. These molecular sizes are in accordance with those reported for
the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast
and cytosolic FBPases underwent increasing inactivation when increasing
concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G
(IgG), respectively, were added to the reaction mixture. However,
inactivations were not observed when the photosynthetic enzyme was
incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative
results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77%
common antigenic determinants between the two chloroplast enzymes when
tested against the spinach photosynthetic FBPase antiserum, which shifted
to 64% when assayed against the pea antiserum. In contrast, common
antigenic determinants between the spinach cytosolic FBPase and the two
chloroplast enzymes were less than 10% when the ELISA test was carried out
with either of the photosynthetic FBPase antisera, and only 5% when the
assay was performed with the antiserum to the spinach cytosolic FBPase.
These results were supported by sequencing data: the deduced amino acid
sequence of a chloroplast FBPase clone isolated from a pea cDNA library
indicated a 39,253 molecular weight protein, with a homology of 85% with
the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme
from spinach.
