Physical and kinetic properties of photosynthetic phosphoenolpyruvate carboxylase in developing apple fruit

Phosphoenolpyruvate carboxylase (PEPC) was partially purified from young developing apple fruit, cultivars Golden Delicious and Cox's Orange Pippin. Freeze-drying of tissue reduced the yield of PEPC activity compared to samples stored at 4°. Activities measured by H¹⁴CO₃⁻ incorporation exceeded the spectrophotometric assay for the enzyme with coupled NADH-malate dehydrogenase (MDH) by up to 60%. The enzyme could be stored at −16° with glycerol and bovine serum albumin for several months without loss of activity. Thermal inactivation of PEPC occurred after heating to 75° for 3 min when MDH was still slightly active. Inhibition of PEPC activity by endogenous phenolics could be prevented by grinding in liquid nitrogen in the presence of polyvinylpyrrolidine and dithiothreitol. Apparent K_m (PEP) and V_max values compared more favourably with those obtained from a C₃-species (spinach) than from a C₄-species (maize). l-Malate (5 mM) inhibited fruit PEPC by 22%; this was decreased to 12% by addition of glucose-6-phosphate (2 mM). From kinetic and effector experiments PEPC in the apple fruit is concluded to be a non-C₄ photosynthetic enzyme.     

Fruit photosynthesis

Abstract. In addition to photosynthesis as in the leaf, fruit possess a system which refixes CO₂ from the mitochondrial respiration of predominantly imported carbon. This pathway produces malate by the action of phosphoenolpyruvate carboxylase, PEPC, (E.C. 4.1.1.31) and appears to be regulated primarily by the cytosolic concentration of HCO₃/CO₂ and malate. Malate is stored in the vacuole as malic acid, constituting a major carbon pool and a potential substrate for respiration. The PEPC in apple fruit proves to be an efficient form of the enzyme with low Michaelis constants, i.e. Km = 0.09 mol m^-3 PEP and 0.2 mol m^–3 HCO₃, and large Ki= 110 mol m^-3 HCO³. In fleshy fruit, chlorophyll and chloroplasts are unevenly distributed; they resemble the C₃ sun-type and arc concentrated in the perivascular tissue, with smaller chloroplasts, fewer grana per chloroplast and a larger degree of vacuolation than commonly found in a leaf of the same species. Fruit photosynthesis often compensates for respiratory CO₂ loss in the light. However, due to respiration in the dark, CO₂ loss is in excess of photosynthetic gain in the light, such that a continual loss of CO₂ was observed in the diurnal cycle and which is maintained throughout fruit development. The rate of CO₂ exchange decreases on a fresh weight or surface basis, but increases with fruit ontogeny on a per fruit basis, causing accumulation of several percent CO₂ in the internal cavity. Stomata are present in the outer epidermis of those fruits examined, but with a 10-to 100-fold lesser frequency than in the abaxial epidermis of leaf of the same species. The number of Stomata is set at anthesis and remained constant, while the stomatal frequency decreases as the fruit surface expands. Stomata are as sensitive as in leaves in the early stages of fruit development, but often are transformed into lenticels during fruit ontogeny, thereby decreasing the permeability of the outer epidermis. The discrepancy between the CO₂-concentrating mechanism provided by PEPC analogous to C₄/CAM Photosynthesis and the kinetics of fruit PEPC, characteristic of C₃/non-autotrophic tissue, suggests the definition of a new type of ‘fruit photosynthesis’ rather than its categorization within an existing type.     

Cold Inactivation of Phosphoenolpyruvate Carboxylase and Pyruvate Orthophosphate Dikinase from the C4 Perennial Plant Atriplex halimus

Phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) cold inactivation was studied in leaf extracts from Atriplex halimus L. Both enzyme activities gradually reduced as the temperature and the total soluble protein decreased. Mg²⁺ at a concentration of 10 mM stabilized PEPC and PPDK activities against cold inactivation. At low Mg²⁺ concentration (4 mM), PEPC was strongly protected by phosphoenolpyruvate, glucose-6-phosphate, and, partially, byL-malate, while PPDK was protected by PEP, but not by its substrate, pyruvate. High concentrations of compatible solutes (glycerol, betaine, proline, sorbitol and trehalose) proved to be good protectants for both enzyme activities against cold inactivation. When illuminated leaves were exposed to low temperature, PPDK was partially inactivated, while the activity of PEPC was not altered.     

Evidence for HCO3− conductance pathways in nutrient membrane of bullfrog antrum

The effect of changing the nutrient-side HCO₃⁻ concentration on potential difference (PD) and resistance in bullfrog antrum bathing in Cl⁻ media was determined. Changes in HCO₃⁻ concentration were from 25 mM to several lower concentrations and back to 25mM. A plot of 6ΔPD6 versus log [HCO₃⁻] gave a linear relation for changes of HCO₃ concentration from 25 down to 3.1 mM and back to 25 mM but deviated to some extent for changes to 1.6 mM. In these experiments, changes from higher to lower HCO₃⁻ concentrations gave a less rapid initial PD response than those in the reverse direction. This result eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part the same changes with a simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of PD decrease was greater by 4.5 mV in the second part. This result indicated that HCO₃⁻ conductance pathways rather than OH⁻ conductance pathways predominated. There was no evidence of HCO₃⁻, OH⁻ and H⁺ conductance pathways in secretory membrane.     

Evidence for HCO3− conductance pathways in nutrient membrane of resting frog fundus

The effect on potential difference (PD) and resistance in Cl⁻ media bathing the resting fundus of Rana pipiens was determined for nutrient HCO₃⁻ changes from 25 mM to several lower concentrations and back to 25 mM. The graph of |vbΔPD|vb versus log[HCO₃⁻] was linear for changes from 25 down to 3.1 mM and also back to 25 mM, but deviated considerably for changes to 1.6 mM. The fact that changes from higher to lower HCO₃⁻ gave a less rapid initial PD response than the reverse direction eliminated H⁺ conductance pathways as being predominant. Experiments were done in which in the first part changes were made in the nutrient solution from 5% CO₂ and 25 mM HCO₃⁻ to 0.6% CO₂ and 3 mM HCO₃⁻ and in the second part, the same changes with the simultaneous change of secretory solution from 5% to 10% CO₂. The magnitude of the PD decrease was greater by 4.0 mV in the second part. This result indicated that HCO₃⁻ rather than OH⁻ conductance pathways predominated. On the secretory side, the change from 25 to 3.1 mM HCO₃⁻ gave a small but significant change in PD. The latter effect was too small to determine whether HCO₃⁻ pathways existed in the secretory membrane.     

Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotrophThiobacillus novellus (ATCC 8093)

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), which catalyzes the carboxylation of phosphoenolpyruvate to produce oxaloacetate was purified 465-fold from extracts of organotrophically grownThiobacillus novellus. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of the purified enzyme revealed the presence of two bands after staining with Buffalo Black. Gels stained with Fast Violet B after incubation with PEP, HCO₃ ^-, Mg²⁺ and acetyl CoA also showed two bands of activity with the faster moving the more active of the two. Sodium dodecylsulfate (SDS)-PAGE of the enzyme heated at 100°C for 5 min revealed the presence of three intensely stained bands of M_r 95 K, 51 K, and 28 K. However, electrophoresis of the enzyme heated for 2 min showed a single band of about 100 K, indicating that the preparation was likely homogeneous. The 51 K and 28 K subunits are thus products of the 95 K subunit. Gel filtration studies of the native enzyme yielded a M_r of 360 K. Therefore, the enzyme is a tetramer. The optimum pH in Tris buffer was 8.0, with K_m for PEP 0.64 mM, HCO₃ ^- 0.11 mM, and acetyl CoA a potent activator, 1.3 M. A divalent cation best served by Mg²⁺ gave sigmoidal initial velocity plots. Hill plots of the data gave coefficients (n_H) of 2.6. None of the metabolites tested, nucleotide triophosphates excepted, significantly affected enzyme activity. Binding studies with¹⁴C-labelled PEP revealed the binding of about 20 moles PEP per mole (360,000 g) of PEPC. Initial velocity studies suggest that the reaction is catalyzed by a random Bi Bi mechanism. Despite the lack of inhibition by certain metabolites, the enzyme's function is probably anaplerotic.Supported by an operating grant from NSERC to AMC.     

Modulation by Bicarbonate of Catalytic and Regulatory Properties of C4Phosphoenolpyruvate Carboxylase from Amaranthus hypochondriacus: Desensitization to Malate and Glucose 6-Phosphate and Sensitization to Mg2+

The catalytic and regulatory properties of phosphoenolpyruvate(PEP) carboxylase (PEPC) are modulated remarkably by the increasein the level of bicarbonate in the assay medium. The activityof PEPC increased by two-fold as the concentration of bicarbonatewas raised from 0.05 to 10 mM. During this state, there wasonly marginal effect on K_m for PEP, while the affinity of PEPCto Mg²⁺ increased by >2 fold. In contrast, the sensitivityof PEPC to malate decreased with increasing concentration ofHCO₃^–. Similarly, the stimulation by glucose 6-phosphate(G-6-P) at optimal concentration (10 mM) of HCO₃^– wasmuch less than that at suboptimal concentration (0.05 mM). K₁for malate increased by about 3 fold and K_a for G-6-P risedby fourfold as bicarbonate concentration was rised from 0.05to 10 mM. These results suggest that HCO₃^– desensitizesPEPC to both malate and G-6-P. Further, these changes were manifestedin both dark- as well as light-forms of the enzyme. Similarresults were obtained with PEPC in leaf extracts or in purifiedform. We therefore propose that bicarbonate-induced changesare independent of phospho-rylation and possibly through a significantchange in the conformation of the enzyme. This is the firstdetailed report indicating marked modulation of regulatory andcatalytic properties of PEPC by bicarbonate, one of its substrate. (Received April 14, 1998; Accepted September 22, 1998)     

Metabolic regulation and gene expression of root phosphoenolpyruvate carboxylase by different nitrogen sources

Alfalfa (Medicago sativa L.) N-sufficient plants were fed 1·5 mM N in the form of NO₃⁻, NH₄⁺ or NO₃⁻ in conjunction with NH₄⁺, or were N-deprived for 2 weeks. The specific activity of phosphoenolpyruvate carboxylase (PEPC) from the non-nodulated roots of N-sufficient plants was increased in comparison with that of N-deprived plants. The PEPC value was highest with NO₃⁻ nutrition, lowest with NH₄⁺ and intermediate in plants that were fed mixed salts. The protein was more abundant in NO₃⁻-fed plants than in either NH₄⁺- or N mixed-fed plants. Nitrogen starvation decreased the level of PEPC mRNA, and nitrate was the N form that most stimulated PEPC gene expression. The malate content was significantly lower in NO₃⁻-deprived than in NO₃⁻-sufficient plants. Root malate accumulation was high in NO₃⁻-fed plants, but decreased significantly in plants that were fed with NH₄⁺. The effect of malate on the desalted enzyme was also investigated. Root PEPC was not very sensitive to malate and PEPC activity was inhibited only by very high concentrations of malate. Asparagine and glutamine enhanced PEPC activity markedly in NO₃⁻-fed plants, but failed to affect plants that were either treated with other N types or N starved. Glutamate and citrate inhibited PEPC activity only at optimal pH. N-nutrition also influenced root nitrate and ammonium accumulation. Nitrate accumulated in the roots of NO₃⁻- and (NO₃⁻ + NH₄⁺)-fed plants, but was undetectable in those administered NH₄⁺. Both the nitrate and the ammonium contents were significantly reduced in NO₃⁻- and (NO₃⁻ + NH₄⁺)-starved plants. Root accumulation of free amino acids was strongly influenced by the type of N administered. It was highest in NH₄⁺-fed plants and the most abundant amides were asparagine and glutamine. It was concluded that root PEPC from alfalfa plants is N regulated and that nitrate exerts a strong influence on the PEPC enzyme by enhancing both PEPC gene expression and activity.     

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure,regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C₄-specific, C₃ or etiolated, CAM and root forms. C₄ leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C₄-dicot Flaveria trinervia. Selective expression of ppc in only C₄-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C₄-PEPC pinpoint the phosphorylatable serine near the N-terminus, C₄-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO₂, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO₃ ^--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C₄ ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C₄ PEPC and the transgenic tobacco plants expressed both C₃ and C₄ isoforms. Site-directed mutagenesis of ppc indicates the importance of His¹³⁸, His⁵⁷⁹ and Arg⁵⁸⁷ in catalysis and/or substrate-binding by the E. coli enzyme, Ser⁸ in the regulation of sorghum PEPC. Important areas for further research on C₄ PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA oxalacetate - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC-protein kinase - PPDK pyruvate, orthophosphate dikinase - Rubisco ribulose 1,5-bis-phosphate carboxylase/oxygenase - CAM Crassulacean acid metabolism     

The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

The ability of the seagrass Zostera muelleri Irmisch ex Aschers. to use HCO₃⁻ as well as CO₂ for photosynthesis was investigated by measuring photosynthetic O₂ evolution over a range of pH values. It was found that the apparent K_m CO₂ fell from 0.128 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO₃⁻ as well as CO₂ may act as a substrate for photosynthesis.The true K_m CO₂ could not be determined due to inhibition of photosynthesis at pHs less than 7.8 K_m CO₂ must be at least 0.128 mM, the apparent K_m at pH 7.9, and is probably of the order of 0.200 mM CO₂, the same as that reported for other marine plants. K_m HCO₃⁻¹ is about 20 mM when CO₂-dependent photosynthesis is minimal. Such a high K_m HCO₃⁻ resembles values reported for freshwater, rather than marine plants.Photosynthetic O₂ evolution is not saturated with respect to total inorganic carbon in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pH 8.1 to 8.5 in the pH profile of photosynthetic O₂ evolution at a constant concentration of inorganic carbon is caused by an effect of pH on HCO₃⁻ uptake. The effect of pH on HCO₃⁻ uptake was determined by constructing a pH profile of photosynthesis at constant HCO₃⁻ concentration, and subtracting the estimated contribution of CO₂ to photosynthesis from this rate. The resultant curve has a maximum at pH 8.4 and declines sharply at pHs less than 8.     

Phosphoenolpyruvate Carboxylase Purified from Leaves of C3, C4, and C3-C4 intermediate species of Alternanthera: Properties at Limiting and Saturating Bicarbonate

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 V_max as well as K_m 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 (V_max), affinity for PEP (K_m), and the sensitivity to malate (K_I) or G-6-P (K_A) 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. This revised version was published online in August 2006 with corrections to the Cover Date.     

Impaired duodenal bicarbonate secretion in diabetic rats. Salutary effect of nitric oxide synthase inhibitor

We previously reported the impaired HCO₃⁻ secretion and the increased mucosal susceptibility to acid in the duodenum of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the salutary effect of the NO synthase inhibitor L-NAME (N^G-nitro-L-arginine methyl ester) on these changes and compared it with those of insulin. Animals were injected streptozotocin (STZ: 70 mg/kg, ip) and used after 1, 3–4, and 5–6 weeks of diabetes with blood glucose levels of > 300 mg/dL. Under urethane anesthesia the HCO₃⁻ secretion was measured in the proximal duodenal loop using a pH-stat method and by adding 10 mM HCl. L-NAME (20 mg/kg × 2) or insulin (4 units/rat) was administered sc for 4–5 weeks, starting 1 week after STZ treatment. The duodenal HCO₃⁻ secretory responses to various stimuli such as mucosal acidification (10 mM HCl for 10 min), 16,16-dimethyl prostaglandin E₂ (dmPGE₂: 10 μg/kg, iv), and vagal stimulation (0.5 mA, 2 ms, 3 Hz) were significantly decreased in STZ-treated rats, depending on the duration of diabetes. Repeated administration of L-NAME, starting from 1 week after STZ treatment, significantly reduced blood glucose levels toward normal values and restored the HCO₃⁻ responses to various stimuli in STZ rats, the effects being similar to those observed after supplementation of insulin. Diabetic rats developed duodenal lesions after perfusion of the duodenum with 150 mM HCl for 4 h, but this ulcerogenic response was significantly inhibited by the repeated treatment with L-NAME as well as insulin. We conclude that L-NAME is effective in ameliorating hyperglycemic conditions in STZ-diabetic rats, similar to insulin, and restores the impaired HCO₃⁻ secretion and the increased mucosal susceptibility to acid in diabetic rat duodenums.     

Stimulatory effect of PACAP on gastroduodenal bicarbonate secretion in rats

The effects of pituitary adenylate cyclase activating polypeptides (PACAPs) on gastroduodenal HCO₃⁻ secretion were investigated in anesthetized rats and compared with those of vasoactive intestinal polypeptide (VIP). Under urethane anesthesia, a rat stomach mounted in an ex vivo chamber (in the absence of acid secretion) or a rat proximal duodenal loop was perfused with saline, and the HCO₃⁻ secretion was measured at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Intravenous injection of PACAP-27 stimulated HCO₃⁻ secretion in a dose-dependent manner in the duodenum but not in the stomach; at 8 nmol/kg PACAP-27 increased the HCO₃⁻ secretion to maximal values of four times greater than basal levels, although this peptide had no effect on duodenal HCO₃⁻ secretion after intracisternal administration (1 nmol/rat). PGE₂ (300 μg/kg, iv) significantly increased HCO₃⁻ secretion in both the stomach and the duodenum. The potency of duodenal HCO₃⁻ secretory action was in the following order; PACAP-27 > PACAP-38 = VIP, and that of PACAP-27 was about 100-fold greater than that of PGE₂. The duodenal HCO₃⁻ secretory action of PACAP-27 as well as PGE₂ was markedly potentiated by prior administration of isobutylmethyl xanthine (10 mg/kg, sc), the inhibitor of phosphodiesterase. Folskolin (250 μg/kg, iv), the stimulator of adenylate cyclase, also increased HCO₃⁻ secretion in the duodenum but not in the stomach. These results suggest that: 1) PACAPs are potent stimulators of HCO₃⁻ secretion in the duodenum but not in the stomach; 2) this action is mediated by cAMP through stimulation of adenylate cyclase; 3) cAMP is a mediator in duodenal but not gastric HCO₃⁻ secretion; and 4) PACAPs may be involved in the peripheral regulation of duodenal HCO₃⁻ secretion.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla verticillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/1) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^?) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from hight Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla vertieillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/l) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^-) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from high Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

Genotypic variability within Tunisian grapevine varieties (Vitis vinifera L.) facing bicarbonate-induced iron deficiency

Morpho-physiological responses to bicarbonate-induced Fe deficiency were investigated in five Vitis vinifera L. Tunisian varieties (Khamri, Blanc3, Arich Dressé, Beldi, and Balta4). One-month-old woody cuttings were cultivated for 85 days on a free calcareous soil irrigated with tap water containing increasing bicarbonate levels (0, 4, 8, 12, and 16 mM NaHCO₃). After this screening, a second experiment compared root biochemical responses of two contrasting genotypes (tolerant-sensitive) dealing with bicarbonate-induced iron deprivation (20 μM Fe ± 10 mM HCO₃⁻) for 75 days. Using morpho-physiological criteria, grapevine tolerance to HCO₃⁻-induced Fe shortage appeared to be genotype-dependent: Balta4 and Beldi varieties showed the highest leaf-chlorosis score (especially at the extreme HCO₃⁻ levels), in contrast to Khamri variety. Growth parameters (shoot height, total leaf area, leaf number, and biomass production) as well as juvenile leaf chlorophyll content were also differently affected depending on both genotype and bicarbonate dose. At 16 mM HCO₃⁻, Khamri was the less sensitive variety, contrasting with Balta4. On the other hand, chlorophyll content correlated positively with HCl-extractible Fe content of the juvenile leaves, suggesting that the grapevine response to iron deficiency may partly depend on to the plant ability to adequately supply young leaves with this element. Root biochemical responses revealed a relatively higher root acidification capacity in Khamri (tolerant) under Fe-deficiency while no significant changes occurred in Balta4 (sensitive). In addition, Fe(III)-reductase and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were strongly stimulated by Fe-deficiency in Khamri, while remaining constant in Balta4. These findings suggest that biochemical parameters may constitute reliable criteria for the selection of tolerant grapevine genotypes to iron chlorosis.     

Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

Effect of sulphite on phosphoenolpyruvate carboxylase and malate formation in extracts of Zea mays

The effect of SO₃^2? on the activity of PEP-carboxylase and on subsequent malate formation has been studied in leaf extracts of Zea mays. PEP-carboxylase was assayed by incorporation of H¹⁴CO₃ - into oxaloacetate dinitrophenylhydrazone and by a spectrophotometric method. In contrast to ribulose diphosphate carboxylase, PEP-carboxylase was not inhibited by 10 mM SO₃^2? with respect to PEP. As was the case with ribulose diphosphate carboxylase, the activity of PEP-carboxylase was inhibited non-competitively with respect to Mg²⁺. However, the K_i value (84.5 mM) was found to be very high. With respect to HCO₃^?, like ribulose diphosphate carboxylase, PEP-carboxylase was inhibited competitively, but the K_i value (27 mM SO₃^2?) increased by about the same factor (× 9) as the K_m, (0·5 mM HCO₃^?) is decreased. This indicates that the replacement of HCO₃^? by SO₃^2?, common to both enzymes, is facilitated by decreasing the affinity of the enzyme for HCO₃^?. At substrate saturating conditions malate formation by the combined action of PEP-carboxylase and endogenous NADH-dependent malate dehydrogenase in leaf extracts was not inhibited by 10 mM SO₃^2?. Although the malate dehydrogenase is inhibited at this SO₃^2? concentration to about 85%, malate formation is unaffected, as PEP-carboxylase is the rate limiting step its turnover rate being only about 8% of NADH-dependent malate dehydrogenase.     

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C₃ or C₄: CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V_max,K_m(PEP)), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4°C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V_max and increased the K_m(PEP) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K_m(PEP) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C₄ species studied, and are consistent with the photosynthetic metabolism of this plant.     

A comparative study of the use of inorganic carbon resources by Chara aspera and Potamogeton pectinatus

We studied the potential role of dissolved inorganic carbon (DIC) in determining vegetation dominance of Potamogeton pectinatus L. and Chara aspera Deth. ex Willd. by monitoring the seasonal dynamics of DIC in a shallow lake and comparing the use of DIC of the two species. The HCO₃⁻-concentration in summer dropped from 2.5 to <0.5 mM with seasonally increasing Chara biomass, whereas outside the vegetation concentrations remained at 2.5 mM. Inside Potamogeton spp. vegetation DIC decreased from 2.5 to ca. 0.75 mM HCO₃⁻. A growth experiment showed ash-free biomass for P. pectinatus was nearly two times as high as for C. aspera at 3 mM HCO₃⁻, but almost two times lower at 0.5 mM than at 3.0. In a separate experiment, P. pectinatus precultured at a relatively low HCO₃⁻-level had a lower net photosynthetic rate (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) than C. aspera (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) over the range of HCO₃⁻-concentrations tested (P_max, 0.14 mmol O₂ g⁻¹ DW h⁻¹). In response to CO₂ no significant differences between the compensation points (P. pectinatus, 28 mM; C. aspera 66 mM), were observed, but the photosynthetic rate increased faster than for C. aspera than for P. pectinatus. Under field conditions, the use of CO₂ is not important since inside vegetation CO₂-concentrations were below 10 μM, and thus, not available for photosynthesis of either species during the main part of the growth season. It is suggested that C. aspera may be a better competitor for HCO₃⁻ than P. pectinatus in conditions with a low HCO₃⁻ supply. As HCO₃⁻ is a strong limiting factor for growth inside the vegetation and probably the only carbon source available, the superior ability of C. aspera to use HCO₃⁻ may be an important factor explaining its present dominance in Veluwemeer.