KINETIC CHARACTERIZATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE EXTRACTEDFROM WHOLE-LEAF AND FROM GUARD-CELL PROTOPLASTS OF VICIA-FABA L (C-3 PLANT) WITH RESPECT TO TISSUE PRE-ILLUMINATION

Whole leaves and guard-cell protoplasts of the C-3 plant Vicia faba L. (broad bean) were separately extracted following a period of illumina tion or following a period of darkness. Kinetic parameters of phosphoe nolpyruvate carboxylase (PEPC, EC 4.1.1.31), V-max and K-m(PEP.Mg), we re determined as a function of assay pH (7.0 or 8.l), the presence of 5 mM glucose-6-P-free (Glc-6-P, an activator), and the presence of 5 m M malate(free) (an inhibitor). On the basis of these parameters, guard -cell PEPC was distinguished from that of whole leaf, indicating eithe r that guard cells contain a unique isoenzyme of PEPC or a different c omplement of isoenzymes or-and less likely-that the obligatorily diffe rent methodologies for the leaf (intact organ) and the guard-cell (pro toplast) enzymes altered them specifically. The values of V-max were r elatively unchanged, regardless of assay conditions or tissue pretreat ment. The values obtained for whole-leaf PEPC V-max were restricted to a small range (52.4 +/- 5.9 (SD) to 64.4 +/- 4.8 (SD) mu mol.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 V-max was also restricted to a small range: 7.48 +/- 0.89 (SD) pm ol.guard-cell pair(-1).h(-1) (pH 8.1, light, +Glc-6-P) to 5.79 +/- 0.6 0 (SD) pmol.guard-cell pair(-1).h(-1) (pH 7.0, dark, +malate). Dependi ng on effectors, and particularly pH, large changes in K-m(PEP.Mg) wer e calculated (whole-leaf PEPC: 0.03 to 3.84 mM; guard-cell PEPC: 0.06 to 3.43 mM). For both extracts, the low values were obtained at pH 8.1 , +Glc-6-P, and the high values at pH 7.0, +malate. Although the range s of K-m values were broadly similar, the PEPCs reacted differently to individual changes in assay components. In very general terms, whole- leaf PEPC was relatively more efficient at pH 8.1, whereas at pH 7.0, the enzymes behaved more similarly. An effect of in vivo pre-illuminat ion on guard-cell PEPC was not detected. A leaf pre-illumination effec t on whole-leaf PEPC was highly statistically significant when assayed under control conditions at pH 7.0. The effect was small - typically a 26% decrease in K-m(PEP.Mg); this typical decrease was less than the range of values in replicate experiments. Such a small pre-illuminati on effect (even it real) could, therefore, easily go undetected. Wheth er such a small change could have physiological relevance is an open q uestion. Neither with the whole-leaf PEPC nor with the guard-cell PEPC was the IC50 (malate) or A(0.5) (Glc-6-P) determined for any conditio n. These kinetic parameters are a focus of present work.