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The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C4 phosphoenolpyruvate carboxylase (PEPC) was investigated with respect to the phosphorylation status of the enzyme. In vitro protein phosphorylation was achieved using the catalytic subunit of a cAMP-dependent protein kinase (PKA) and a recombinant, immunopurified PEPC (0.9 mol of covalent Pi/mol PEPC subunit). Between pH 6.8 and 8, velocity and IC50 for L-malate increased for both the nonphosphorylated and the phosphorylated forms. With respect to the nonphosphorylated PEPC, the phospho-PEPC always gave high values for these kinetic parameters at the pH range investigated, especially between pH 7 and 7.3. The phosphorylation-induced stimulation of PEPC activity was four- to fivefold at pH 7.1 and approximately twofold at pH 7.3. The IC50 for L-malate showed a two- to threefold increase at pH 7.3, but varied less at pH 7.1 upon PEPC phosphorylation. Thus, phosphorylation of PEPC caused a predominant V effect or a mixed (V/IC50) effect at pH 7.1 or 7.3, respectively. This was also observed with the enzyme from desalted crude protein extracts from dark or light-adapted Sorghum leaves and leaf-derived mesophyll protoplasts illuminated in the presence of methylamine, a compound known to increase cytosolic pH (pHc). At pH 7.3, desensitization to L-malate pf phospho-PEPC was due to an enhanced ability of PEP to compete with the inhibitor. The positive effector glucose-6P acted similarly to phosphorylation; however a combination of both factors (glucose-6P and phosphorylation) led to a much larger increase in the IC50 for L-malate than that observed by a single factor. These effects may account for the actual CO2 assimilation rate of an illuminated Sorghum leaf. In reconstituted assays performed at pH 7.1 or 7.3, in the presence of PKA, the addition of 10 mM L-malate decreased the phosphorylation rate of the PEPC. This effect was strongly inhibited by the addition of glucose-6P (5 mM). It is suggested that modulation of the phosphorylation rate is partly through conformational changes in the PEPC induced by these photosynthetic metabolites. Together with previous findings indicating that the photoinduction of PEPC protein kinase was strictly dependent on cytosolic alkalization in meosphyll cell protoplasts (J. N. Pierre et al., 1992, Eur. J. Biochem. 210, 531-537), the present data support the view that cytosolic pH is an important component of the overall regulation of PEPC in C4-photosynthesis. Finally, the role of phosphorylation appears to be a mechanism to amplify the regulatory influences of pHc and glucose-6P on PEPC activity.

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