Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize

The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K_{0.5 PEP} for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K_{0.5 PEP}, while the K_{0.5 PEP} of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme’s kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.     

Changes in Properties of Phosphoenolpyruvate Carboxylase with Induction of Crassulacean Acid Metabolism (CAM) in the C4 Plant Portulaca Oleracea

Aiming at understanding the odd case of CAM expression by a C₄ plant, some properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, orthophosphate: oxaloacetate carboxylyase, phosphorylating) were comparatively studied in leaves of CAM-expressing and non-expressing Portulaca oleracea L. plants. CAM expression was induced by growing plants under an 8-h photoperiod and under water-stress. CAM induction in leaves of these plants (designated as CAM) is indicated by the nocturnal acidification and by the clear diurnal oscillation pattern and amplitude of acidity, malic acid, and PEPC activity characteristic of CAM plants. Treatment of the other plant group (designated as C₄) by growth under a 16-h photoperiod and well-watered conditions did not induce expression of the tested criteria of CAM in plants. In these C₄ plants, the mentioned CAM criteria were undetectable. PEPC from CAM and C₄ Portulaca responded differently to any of the studied assay conditions or effectors. For example, extent and timing of sensitivity of PEPC to pH change, inhibition by malate, activation by glucose-6-phosphate or inorganic phosphate, and the enzyme affinity to the substrate PEP were reversed with induction of CAM from the C₄-P. oleracea. These contrasting responses indicate distinct kinetic and regulatory properties of PEPC of the two modes. Thus by shifting to CAM in the C₄ Portulaca a new PEPC isoform may be synthesised to meet CAM requirements. Simultaneous occurrence of both C₄ and CAM is suggested in P. oleracea when challenged with growth under stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Light modulation of glucose-6-phosphate dehydrogenase: partial characterization of the light inactivation system and its effects on the properties of the chloroplastic and cytoplasmic forms of the enzyme

Light inactivation of glucose-6-phosphate dehydrogenase within the pea (Pisum sativum L.) leaf chloroplast has a narrow pH optimum between 7.2 and 7.4 and is NADP-sensitive. The pH optimum for dark activation is slightly lower. Inactivation apparently results in a simple decrease in maximal velocity of the chloroplastic and cytoplasmic forms of the enzyme with no concomitant change in pH optimum or K_m (glucose 6-phosphate).     

Acid phosphatase from maize scutellum: Negative cooperativity suppression by glucose

Acid phosphatase purified from maize scutellum, upon acylation with succinic anhydride, still shows negative co-operativity for the hydrolysis of glucose-6-phosphate at pH 5.4. This phenomenon is abolished by glucose, for both native and succinylated enzymes, through stimulation of the initial velocities at sub-optimal substrate concentrations. However, negative co-operativity for the enzymatic hydrolysis of p-nitrophenylphosphate at pH 5.4 is suppressed only at high concentrations of glucose. Furthermore, the hydrolysis of p-nitrophenylphosphate is noncompetitively inhibited (low affinity form of the enzyme molecule) by glucose, which suggests the existence of different substrate binding sites.     

Diurnal regulation of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase appears to be located in or associated with the chloroplasts of Crassula. As has been found with this enzyme in other CAM plants, a crude extract of leaves gathered during darkness and rapidly assayed for phosphoenolpyruvate carboxylase (PEPc) activity is relatively insensitive to inhibition by malate. After illumination begins, the PEPc activity becomes progressively more sensitive to malate. This enzyme also shows a diurnal change in activation by glucose-6-phosphate, with the enzyme from dark leaves more strongly activated than that from leaves in the light.

When the enzyme is partially purified in the presence of malate, the characteristic sensitivity of the day leaf enzyme is largely retained. Partial purification of the enzyme from dark leaves results in a small increase in sensitivity to malate inhibition.

Partially purified enzyme is found by polyacrylamide gel electrophoresis analysis to have two bands of PEPc activity. In enzymes from dark leaves, the slower moving band predominates, but in the light, the faster moving band is preponderant. Both of these bands are shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be composed of the same subunit of 103,000 daltons.

The enzyme partially purified from night leaves has a pH optimum of 5.6, and is relatively insensitive to malate inhibition over the range from pH 4.5 to 8. The enzyme from day leaves has a pH optimum of 6.6 and is strongly inhibited by malate at pH values below 7, but becomes insensitive at higher pH values.

Gel filtration of partially purified PEPc showed two activity peaks, one corresponding approximately to a dimer of the single subunit, and the other twice as large. The larger protein was relatively insensitive to malate inhibition, the smaller was strongly inhibited by malate.

Kinetic studies showed that malate is a mixed type inhibitor of the sensitive, day, enzyme, increasing K_m for phosphoenolpyruvate and reducing V_max. With the insensitive, night, enzyme, malate is a K type inhibitor, reducing the K_m for phosphoenolpyruvate, but having little effect on V_max. The inhibition of the insensitive enzyme by malate appears to be hysteretic, taking several minutes to be expressed during assay, probably indicating a change in the conformation or aggregation state of the enzyme.

Activation by glucose-6-phosphate is of the mixed type for the day form of the enzyme, causing both a decreased K_m for phosphoenolpyruvate and an increased V_max, but the night, or insensitive, form shows only an increase in V_max in response to glucose-6-phosphate.

     

Diurnal changes in the regulatory properties of PEP-carboxylase in Crassulacean Acid Metabolism (CAM)

Abstract. The CAM plants Kalanchoe tubiflora and K. blossfeldiana were grown under photoperiodically controlled conditions (short days). In these plants, phos-phoenolpyruvate carboxylase capacity and the sensitivity of the enzyme to the effectors L-malate (inhibitor) and glucose-6-phosphate (activator) were measured throughout the diurnal CAM cycle. In K. tubiflora , enzyme capacity was higher if measured at pH 7.0 than at pH 8.0 and displayed a rhythmical behavior with highest values at the end of the light period. As reported earlier, in K. blossfeldiana PEP-C capacity was higher during the night. It was more pronounced when plants were kept in CO₂-free air during the dark period. In both plants, the sensitivity of the enzyme to the effectors showed very clear diurnal changes: inhibition by malate and activation by glucose-6-phosphate were strikingly higher during the day than during the night; the effect depended on PEP concentration. The changing activation of the enzyme by glucose-6-phos-phate reflects diurnal changes of the Km for PEP which was found to be higher during the day than during the night. Manipulations of malate accumulation by nocturnal application of CO₂-free air did not influence these effects. The results are discussed in context with the metabolic control of CAM.     

Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases

The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg²⁺, ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.     

Shifts in the Carbon Metabolism of Xerosicyos danguyi H. Humb. (Cucurbitaceae) Brought About by Water Stress : II. Enzymology

Xerosicyos danguyi Humbert (Cucurbitaceae) is a leaf succulent endemic to Madagascar. Under well-watered conditions, the plant exhibited Crassulacean acid metabolism (CAM) but shifted to a dampened form of CAM, CAM-idling, when subjected to water stress. The purpose of this investigation was to examine the effects of a shift in carbon metabolism on phosphoenolpyruvate carboxylase and on NADP-malic enzyme in X. danguyi. Experiments were conducted to determine the diurnal patterns of enzyme activity and pH optima of both enzymes, as well as the approximate molecular mass, kinetic patterns, malate inhibition, and glucose-6-phosphate stimulation of phosphoenolpyruvate carboxylase. The two enzymes extracted from well-watered and water-stressed plants were similar in most parameters investigated; thus, CAM-idling appeared to be only a dampened form of CAM photosynthesis.     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C(3), C(4), and Crassulacean Acid Metabolism Plants

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C₃ plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C₄ plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C₄ plants, 0.5% in the CAM plant, and less than 0.01% in C₃ plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C₃ and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C₃ and CAM plants than to those of C₄ plants.     

Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was purified 40-fold from soybean (Glycine max L. Merr.) nodules to a specific activity of 5.2 units per milligram per protein and an estimated purity of 28%. Native and subunit molecular masses were determined to be 440 and 100 kilodaltons, respectively, indicating that the enzyme is a homotetramer. The response of enzyme activity to phosphoenolpyruvate (PEP) concentration and to various effectors was influenced by assay pH and glycerol addition to the assay. At pH 7 in the absence of glycerol, the K_m (PEP) was about twofold greater than at pH 7 in the presence of glycerol or at pH 8. At pH 7 or pH 8 the K_m (MgPEP) was found to be significantly lower than the respective K_m (PEP) values. Glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, and dihydroxyacetone phosphate activated PEPC at pH 7 in the absence of glycerol, but had no effect under the other assay conditions. Malate, aspartate, glutamate, citrate, and 2-oxoglutarate were potent inhibitors of PEPC at pH 7 in the absence of glycerol, but their effectiveness was decreased by raising the pH to 8 and/or by adding glycerol. In contrast, 3-phosphoglycerate and 2-phosphoglycerate were less effective inhibitors at pH 7 in the absence of glycerol than under the other assay conditions. Inorganic phosphate (up to 20 millimolar) was an activator at pH 7 in the absence of glycerol but an inhibitor under the other assay conditions. The possible significance of metabolite regulation of PEPC is discussed in relation to the proposed functions of this enzyme in legume nodule metabolism.     

Metabolite activation of crassulacean Acid metabolism and c(4) phosphoenolpyruvate carboxylase

The effects of glycine, alanine, serine, and various phosphorylated metabolites on the activity of phosphoenolpyruvate (PEP) carboxylase from Zea mays and Crassula argentea were studied. The maize enzyme was found to be activated by amino acids at a site that is separate from the glucose 6-phosphate binding site. The combination of glycine and glucose 6-phosphate synergistically reduced the apparent K_m of the enzyme for PEP and increased the apparent V_max. Of the amino acids tested, glycine showed the lowest apparent K_a and caused the greatest activation. d-Isomers of alanine and serine were more effective activators than the l-isomers. Unlike the maize enzyme, the Crassula enzyme was not activated by amino acids. Activation of either the Crassula or maize enzyme by glucose 6-phosphate occurred without dephosphorylation of the activator molecule. Furthermore, the Crassula enzyme was activated by two compounds containing phosphonate groups whose carbon-phosphorus bonds were not cleaved by the enzyme. A study of analogs of glucose 6-phosphate with Crassula PEP carboxylase revealed that the identity of the ring heteroatom was a significant structural feature affecting activation. Activation was not highly sensitive to the orientation of the hydroxyl group at the second or fourth carbon positions or to the presence of a hydroxyl group at the second position. However, the position of the phosphate group was found to be a significant factor.     

Regulation of mitochondrial NAD-malic enzyme involved in C4 pathway photosynthesis.

Mitochondrial NAD-malic enzyme isolated from bundle sheath cells of different C₄ species was activated 5- to 15-fold by fructose 1,6-phosphate. With 2.5 mm malate, fructose 1,6-phosphate was optimally active between 30 and 100 μm and activation was similar to that previously reported for CoA and acetyl-CoA. 3-Phosphoglycerate and isocitrate were less effective activators and other metabolites including fructose 6-phosphate and glyceraldehyde 3-phosphate were without effect. Depending on the source of the enzyme, the response to increasing fructose 1,6-phosphate was either sigmoidal, with activation resulting from an increase in the affinity of the enzyme for malate, or hyperbolic, in which case the activator increased maximum velocity. Bicarbonate inhibited NAD-malic enzyme activity, acting competitively with respect to both malate and the activators fructose 1,6-phosphate and CoA. The enzyme was also inhibited in a similar competitive manner by higher concentrations of chloride and nitrate ions. Decarboxylation of C₄ acids by isolated mitochondria was inhibited by bicarbonate and nitrate ions, and, as with isolated NAD-malic enzyme, inhibition was competitive with respect to malate. The rate of C₄ acid decarboxylation by freshly prepared mitochondria was not increased by adding fructose 1,6-phosphate or CoA. However, decarboxylating activity declined after incubating mitochondria at 30 °C without C₄ acids, and this loss of activity was largely prevented by fructose 1,6-phosphate. Mitochondria were found to decarboxylate oxaloacetate as rapidly as aspartate.     

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.     

Inhibition by Catalase of Dark-mediated Glucose-6-Phosphate Dehydrogenase Activation in Pea Chloroplasts

Dark activation of light-inactivated glucose-6-phosphate dehydrogenase was inhibited by catalase in a broken pea chloroplast system. Partially purified glucose-6-phosphate dehydrogenase from pea leaf chloroplasts can be inactivated in vitro by dithiothreitol and thioredoxin and reactivated by H₂O₂. The in vitro activation by H₂O₂ was not enhanced by horseradish peroxidase, and dark activation in the broken chloroplast system was only slightly inhibited by NaCN. These results indicate that the dark activation of glucose-6-phosphate dehydrogenase may involve oxidation by H₂O₂ of SH groups on the enzyme which were reduced in the light by the light effect mediator system.     

Light activation of maize phosphoenolpyruvate carboxylase protein-serine kinase activity is inhibited by mesophyll and bundle sheath-directed photosynthesis inhibitors

C₄ phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC-PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4-dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (dl-glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays L.) PEPC-PK and the ensuing regulatory phosphorylation of its target enzyme in vivo. Based on these and related observations, we propose that the Calvin cycle supplies the C₄ mesophyll cell with (a) a putative signal (e.g. phosphorylated metabolite, amino acid) that interacts with the cytoplasmic protein synthesis event to effect the light activation of PEPC-PK and the concomitant phosphorylation of PEPC, and (b) high levels of known positive effectors (e.g. triose-phosphate, glucose-6-phosphate) that interact directly with the carboxylase. The combined result of this complex regulatory cascade is to effectively desensitize PEPC to feedback inhibition by the millimolar levels of l-malate required for rapid diffusive transport to the bundle sheath during high rates of C₄ photosynthesis.     

Regulation of C4-phosphoenolpyruvate carboxylase activity by ambient CO2

Light activation of phosphoenolpyruvate carboxylase from the leaves of the C₄ plant Setaria verticillata (L.) is more pronounced at low CO₂ levels. The 2-fold activation observed at physiological ambient CO₂ becomes 3.64-fold at 5 L/L and completely abolished above 700 L/L. When the stomata close under the influence of abscisic acid at 330 L/L CO₂, the extent of light activation is high (3.59-fold), probably because the increased diffusive resistance keeps the internal CO₂ at much lower levels. Under darkness. CO₂ and absicisic acid do not affect the extractable phosphoenolpyruvate carboxylase activity. Internal CO₂ levels may determine phosphoenolpyruvate concentratio in the cytoplasm through the control of its utilization by phosphoenolpyruvate carboxylase. We have recently proposed (Samaras et al. 1988) that photosynthetically produced phosphoenolpyruvate could be an activator of the enzyme. It is therefore suggested that CO₂ indirectly affects the activation state of phosphoenolpyruvate carboxylase by controlling the levels of phosphoenolpyruvate which may act as an activator.Abbreviations PEPCase phosphoenolpyruvate carboxylase - PEP phosphoenolpyruvate - PAR photosynthetically active radiation - G6P glucose-6-phosphate - ABA abscisic acid - MDH malate dehydrogenase - PPDK pyruvate, Pi, dikinase - CAM Crassulacean Acid Metabolism     

Patterns of phosphoenolpyruvate carboxylase activity and cytosolic pH during light activation and dark deactivation in C3 and C4 plants

The rate and extent of light activation of PEPC may be used as another criterion to distinguish C₃ and C₄ plants. Light stimulated phosphoenolypyruvate carboxylase (PEPC) in leaf discs of C₄ plants, the activity being three times greater than that in the dark but stimulation of PEPC was limited about 30% over the dark-control in C₃ species. The light activation of PEPC in leaves of C₃ plants was complete within 10 min, while maximum activation in C₄ plants required illumination for more than 20 min, indicating that the relative pace of PEPC activation was slower in C₄ plants than in C₃ plants. Similarly, the dark-deactivation of the enzyme was also slower in leaves of C₄ than in C₃ species. The extent of PEPC stimulation in the alkaline pH range indicated that the dark-adapted form of the C₄ enzyme is very sensitive to changes in pH. The pH of cytosol-enriched cell sap extracted from illuminated leaves of C₄ plants was more alkaline than that of dark-adapted leaves. The extent of such light-dependent alkalization of cell sap was three times higher in C₄ leaves than in C₃ plants. The course of light-induced alkalization and dark-acidification of cytosol-enriched cell sap was markedly similar to the pattern of light activation and dark-deactivation of PEPC in Alternanthera pungens, a C₄ plant. Our report provides preliminary evidence that the photoactivation of PEPC in C₄ plants may be mediated at least partially by the modulation of cytosolic pH.Abbreviations CAM Crassulacean acid metabolism - G-6-P glucose-6-phosphate - PMSF phenylmethylsulfonyl fluoride - PEPC phosphoenolpyruvate carboxylase - PEPC-PK phosphoenolpyruvate ca carboxylase-protein kinase     

Regulation of mammalian hexokinase: regulatory differences between isoenzyme I and II

Mammalian hexokinase isoenzymes I and II have been shown to differ qualitatively in response to various modifiers. Although both enzymes are inhibited by glucose 6-phosphate, only isoenzyme II exhibits a slow response to the presence of this inhibitor. P_i decreases the affinity of glucose 6-phosphate for Sarcoma 37 hexokinase I, but has no effect on hexokinase II from the same cell. P_i overcomes all of the inhibition of red cell hexokinase by glucose-6-P and hence the two effectors act competitively. At pH 6.5, catecholamines increase the V of isoenzyme I of Sarcoma 37 and brain in the soluble and mitochondrial forms but do not activate these forms of tumor isoenzyme II. Citrate activates brain and tumor isoenzyme I when they are inhibited by tris(hydroxy-methyl)aminomethylethane sulfonate (TES) and ADP; however, tumor isoenzyme II is not activated.