PLANT PHYSIOLOGY , Vol 103, Issue 4 1189-1194, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
The Interactive Effects of pH, L-Malate, and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase
M. C. Tarczynski and W. H. Outlaw Jr
Department of Biological Science, Florida State University, Tallahassee, Florida 32306-3050
The interactive effects of pH, L-malate, and glucose-6-phosphate (Glc-6-P)
on the Vmax and Km of guard-cell (GC) phosphoenolpyruvate (PEP) carboxylase
(PEPC) of Vicia faba L. were determined. Leaves of three different
physiological states (closed stomata, opening stomata, open stomata) were
rapidly frozen and freeze dried. GC pairs dissected from the leaves were
individually extracted and individually assayed for the kinetic properties
of PEPC. Vmax was 6 to 9 pmol GC pair-1 h-1 and was apparently unaffected
to a biologically significant extent by the investigated physiological
states of the leaf, pH (7.0 or 8.5), L-malate (0, 5, or 15 mM), and Glc-6-P
(0, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the Km(PEP.Mg) was about
0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a GC [PEP]
of 0.27 mM. In the study reported here, we determined that the in situ GC
[Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Glc-6-P was
included in the GC PEPC assay mixture, the Km(PEP.Mg) decreased to about
0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Glc-6-P at endogenous
concentrations would seem both to activate the enzyme and to diminish the
dramatic effect of pH on Km(PEP.Mg). Under assay conditions, L-malate is an
inhibitor of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8
mM. Inclusion of 5 mM L-malate increased the Km(PEP.Mg) to about 3.6 mM (pH
7.0) or 0.4 mM (pH 8.5). Glc-6-P (0.5 mM) was sufficient to relieve
L-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM
Glc-6-P was required to relieve L-malate inhibition at pH 7.0. No
biologically significant effect of physiological state of the tissue on GC
PEPC Km(PEP.Mg) (regardless of the presence of effectors) was observed.
Together, these results are consistent with a model that GC PEPC is
regulated by its cytosolic chemical environment and not by
posttranslational modification that is detectable at physiological levels
of effectors. It is important to note, however, that we did not determine
the phosphorylation status of GC PEPC directly or indirectly (by comparison
of the concentration of L-malate that causes a 50% inhibition of GC PEPC).
