Feeding K+ or Na+ nitrate salts in vivo enhanced the activity of phosphoenolpyruvate carboxylase (PEPC) in the leaf extracts of Alternanthera pungens (C4 plant) and A. sessilis (C3 species). The increase was more pronounced in A. pungens than in A. sessilis. Chloride salts increased the PEPC activity only marginally. However, the sulfate salts were either not effective or inhibitory. Feeding nitrate modulated the regulatory properties of PEPC in A. pungens, resulting in increased KI (malate) and decreased KA (glucose-6-P). The sensitivity of PEPC to malate, which gives a measure of phosphorylation status of the enzyme, indicated that feeding leaves with NO3- enhanced the phosphorylation status of the enzyme. The reduction in PEPC activity due to cycloheximide treatment suggested that increased synthesis of PEPC protein kinase may be one of the reasons for the enhancement in PEPC activity, after the nitrate feeding. We suggest that nitrate salts could be used as a tool to modulate and analyze the properties of PEPC in C3 and C4 plants. and A. V. Rajagopalan, R. M. Agarwal, A. S. Raghavendra.
Phosphoenolpyruvate carboxylase (PEPC) was purified from leaves of four species of Alternanthera differing in their photosynthetic carbon metabolism: Alternanthera sessilis (C3), A. pungens (C4), A. ficoides and A. tenella (C3-C4 intermediates or C3-C4). The activity and properties of PEPC were examined at limiting (0.05 mM) or saturating (10 mM) bicarbonate concentrations. The Vmax as well as Km values (for Mg2+ or PEP) of PEPC from A. ficoides and A. tenella (C3-C4 intermediates) were in between those of C3 (A. sessilis) and C4 species (A. pungens). Similarly, the sensitivity of PEPC to malate (an inhibitor) or G-6-P (an activator) of A. ficoides and A. tenella (C3-C4) was also of intermediate status between those of C3 and C4 species of A. sessilis and A. pungens, respectively. In all the four species, the maximal activity (Vmax), affinity for PEP (Km), and the sensitivity to malate (KI) or G-6-P (KA) of PEPC were higher at 10 mM bicarbonate than at 0.05 mM bicarbonate. Again, the sensitivity to bicarbonate of PEPC from C3-C4 intermediates was in between those of C3- and C4-species. Thus the characteristics of PEPC of C3-C4 intermediate species of Alternanthera are intermediate between C3- and C4-type, in both their kinetic and regulatory properties. Bicarbonate could be an important modulator of PEPC, particularly in C4 plants. and Bhaskarrao Chinthapalli ... [et al.].
A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg-1(protein) s-1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40-60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, -20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen. and J. Gayathri, K. Parvathi, A. S. Raghavendra.