THE INTERACTIVE EFFECTS OF PH, L-MALATE, AND GLUCOSE-6-PHOSPHATE ON GUARD-CELL PHOSPHOENOLPYRUVATE CARBOXYLASE

The interactive effects of pH, L-Malate, and glucose-6-phosphate (Glc- 6-P) on the V(max) and K(m) of guard-cell (GC) phosphoenolpyruvate (PE P) carboxylase (PEPC) of Vicia faba L. were determined. Leaves of thre e different physiological states (closed stomata, opening stomata, ope n stomata) were rapidly frozen and freeze dried. GC pairs dissected fr om the leaves were individually extracted and individually assayed for the kinetic properties of PEPC. V(max) 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 r eported earlier, the K(m(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 stu dy reported here, we determined that the in situ GC [Glc-6-P] equals a pproximately 0.6 to 1.2 mm. When 0.5 mm Glc-6-P was included in the GC PEPC assay mixture, the K(m(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 woul d seem both to activate the enzyme and to diminish the dramatic effect of pH on K(m(PEP.Mg)). Under assay conditions, L-malate is an inhibit or of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8 mm . Inclusion of 5 mM L-malate increased the K(m(PEP.Mg)) to about 3.6 m m (pH 7.0) or 0.4 mm (pH 8.5). Glc-6-P (0.5 mm) was sufficient to reli eve L-malate inhibition completely at pH 8.5. In contrast, approximate ly 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 tiss ue on GC PEPC K(m(PEP.Mg)) (regardless of the presence of effectors) w as 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 lev els of effectors. It is important to note, however, that we did not de termine the phosphorylation status of GC PEPC directly or indirectly ( by comparison of the concentration Of L-malate that causes a 50% inhib ition of GC PEPC).