Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination.

Whole leaves and guard-cell protoplasts of the C3 plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), Vmax and Km(PEP.Mg), were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mM glucose-6-Pfree (Glc-6-P, an activator), and the presence of 5 mM malatefree (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or--and less likely--that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically. The values of Vmax were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC Vmax were restricted to a small range (52.4 +/- 5.9 (SD) to 64.4 +/- 4.8 (SD) mumol.g fresh mass-1.h-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC Vmax was also restricted to a small range: 7.48 +/- 0.89 (SD) pmol.guard-cell pair-1.h-1 (pH 8.1, light, +Glc-6-P) to 5.79 +/- 0.60 (SD) pmol.guard-cell pair-1.h-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in Km(PEP.Mg) were calculated (whole-leaf PEPC: 0.03 to 3.84 mM; guard-cell PEPC: 0.06 to 3.43 mM).(ABSTRACT TRUNCATED AT 250 WORDS)