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
✍ Scribed by Wang, Xue-Chen ;Outlaw, William H. ;De Bedout, Jean André ;Du, Zhirong
- Publisher
- Springer
- Year
- 1994
- Tongue
- English
- Weight
- 970 KB
- Volume
- 26
- Category
- Article
- ISSN
- 0018-2214
No coin nor oath required. For personal study only.
✦ Synopsis
Whole leaves and guard-cell protoplasts of the C 3 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), V~a x and K~Ep.~g ~, were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mM glucose-6-P~ e (Glc-6-P, an activator), and the presence of 5 mM malat%e e (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 Vma x were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC Vr~x were restricted to a small range (52.4 q-5.9 (SD) to 64.4 + 4.8 (SD) p, mol -g flesh mass -~. h ~; 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 Vm~ was also restricted to a small range: 7.48 4-0.89 (SD) pmol. guard-cell pair 1. h ~ (pH 8.1, light, +Glc-6-P) to 5.79 4-0.60 (SD) pmol" guard-cell pair -I. h -1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH large changes in Kmp~p M were ( 9 g) calculated (whole-leaf PEPC: 0.03 to 3.84 raM; guard-cell PEPC: 0.06 to 3.43 raM). 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 ranges 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-illumination on guard-cell PEPC was not detected. A leaf pre-illumination effect 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 ; this typical decrease was less than the range of values in replicate experiments. Such a small pre-illumination effect (even~r~al) could, therefore, easily go undetected. Whether such a small change could have physiological relevance is an open question. Neither with the whole-leaf PEPC nor with the guard-cell PEPC was the ICs0 (malate) or A0. 5 (Glc-6-P) determined for any condition. These kinetic parameters are a focus of present work.