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1.
The effects of adenine nucleotides on phosphoenolypyruvate carboxylase were investigated using purified enzyme from the CAM plant, Crassula argentea. At 1 millimolar total concentration and with limiting phosphoenolpyruvate, AMP had a stimulatory effect, lowering the Km for phosphoenolpyruvate, ADP caused less stimulation, and ATP decreased the activity by increasing the Km for phosphoenolpyruvate. Activation by AMP was not additive to the stimulation by glucose 6-phosphate. Furthermore, AMP increased the Ka for glucose 6-phosphate. Inhibition by ATP was competitive with phosphoenolpyruvate. In support of the kinetic data, fluorescence binding studies indicated that ATP had a stronger effect than AMP on phosphoenolpyruvate binding, while AMP was more efficient in reducing glucose 6-phosphate binding. As free Mg 2+ was held constant and saturating, these effects cannot be ascribed to Mg 2+ chelation. Accordingly, the enzyme response to the adenylate energy charge was basically of the “R” type (involving enzymes of ATP regenerating sequences) according to D. E. Atkinson's (1968 Biochemistry 7: 4030-4034) concept of energy charge regulation. The effect of energy charge was abolished by 1 millimolar glucose 6-phosphate. Levels of glucose 6-phosphate and of other putative regulatory compounds of phosphoenolpyruvate carboxylase were determined in total leaf extracts during a day-night cycle. The level of glucose 6-phosphate rose at night and dropped sharply during the day. Such a decrease in glucose 6-phosphate concentration could permit an increased control of phosphoenolpyruvate carboxylase by energy charge during the day. 相似文献
2.
In vivo CO 2 fixation and in vitro phosphoenolpyruvate (PEP) carboxylase levels have been measured in lupin ( Lupinus angustifolius L.) root nodules of various ages. Both activities were greater in nodule tissue than in either primary or secondary root tissue, and increased about 3-fold with the onset of N 2 fixation. PEP carboxylase activity was predominantly located in the bacteroid-containing zone of mature nodules, but purified bacteroids contained no activity. Partially purified PEP carboxylases from nodules, roots, and leaves were identical in a number of kinetic parameters. Both in vivo CO 2 fixation activity and in vitro PEP carboxylase activity were significantly correlated with nodule acetylene reduction activity during nodule development. The maximum rate of in vivo CO 2 fixation in mature nodules was 7.9 nmol hour −1 mg fresh weight −1, similar to rates of N 2 fixation and reported values for amino acid translocation. 相似文献
3.
Metal ion interactions with phosphoenolpyruvate carboxylase from the CAM plant Crassula argentea and the C 4 plant Zea mays were kinetically analyzed. Fe 2+ and Cd 2+ were found to be active metal cofactors along with the previously known active metals Mg 2+, Mn 2+, and Co 2+. In studies with the Crassula enzyme, Mg 2+ yielded the highest Vmax value but also generated the highest values of Km(metal) and Km(pep). For these five active metals lower Km(metal) values tended to be associated with lower Km(pep) values. PEP saturation curves showed more kinetic cooperativity than the corresponding metal saturation curves. The activating metal ions all have ionic radii in the range of 0.86 to 1.09 Å. Ca 2+, Sr 2+, Ba 2+, and Ni 2+ inhibited competitively with respect to Mg 2+, whereas Be 2+, Cu 2+, Zn 2+, and Pd 2+ showed mixed-type inhibition. Vmax trends with the five active metals were similar for the C. argentea and Z. mays enzymes except that Cd 2+ was less effective with the maize enzyme. Km(metal) values were 10- to 60-fold higher in the enzyme from Z. mays. 相似文献
4.
The pathway of autotrophic CO 2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO 2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO 2 or [ 14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO 2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus. 相似文献
5.
Phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to light in C 3 and C 4 plants and to a circadian oscillator in CAM plants. Increases in phosphoenolpyruvate carboxylase kinase activity require protein synthesis. This requirement has been analysed by quantifying translatable mRNA for this protein kinase using in vitro translation of isolated RNA followed by direct assay of kinase activity. In leaves of the CAM plant Bryophyllum (Kalanchoë) fedtschenkoi, in normal diurnal conditions, kinase mRNA was 20-fold more abundant at night than in the day. In constant environmental conditions (continuous darkness, CO 2-free air, 15°C) kinase mRNA exhibited circadian oscillations. The circadian disappearance of kinase mRNA and kinase activity was delayed by lowering the temperature to 4°C and accelerated by raising the temperature to 30°C. The appearance of kinase mRNA and activity was blocked by cordycepin and puromycin. In maize and barley, kinase mRNA increased in response to light. For all three plants, the phosphoenolpyruvate carboxylase kinase activity generated during in vitro translation was Ca 2+-independent. These results demonstrate that phosphoenolpyruvate carboxylase kinase activity is regulated at the level of translatable mRNA in C 3, C 4 and CAM plants. 相似文献
6.
In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K
m
values for PEP and NaHCO 3 were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K
m
for Mn 2+, Co 2+ and Mg 2+ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg 2+. Mn 2+ and Co 2+ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA ( K
a
0.18 mM) or propionyl CoA ( K
a
0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP
phosphoenolpyruvate
- OAA
oxaloacetate
- MW
molecular weight
- TEMG buffer
50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl 2, 1 mM glutathione
- HEPES
N-2-hydroxyethylpiperazine-N-ethanesulfonic acid 相似文献
7.
To determine enzymatic activities in the thermotolerant strain K1 (formerly Sulfobacillus thermosulfidooxidans subsp. thermotolerans), it was grown in a mineral medium with (1) thiosulfate and Fe 2+ or pyrite (autotrophic conditions), (2) Fe 2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO 2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO 2 in the medium. 相似文献
8.
Spinach leaf phosphoenolpyruvate carboxylase has been purified to homogeneity using salt fractionatjon, chromatography, and immunologie procedures to remove contaminating ribulose diphosphate carboxylase. From gel filtration and isoelectric focusing, the molecular weight (~560,000) and isoelectric point (p I = 4.9) are indistinguishable from those of ribulose diphosphate carboxylase. The subunit molecular weight of phosphoenolpyruvate carboxylase (130,000) suggests that the native enzyme is a tetramer.Kinetic studies using Mg 2+ or Mn 2+ as the activator indicate that the divalent cation lowers the Km of the substrate phosphoenolpyruvate by an order of magnitude and conversely, that the presence of the substrate similarly lowers the Km of the metal ion, suggesting an enzyme-metal-substrate bridge complex. Three analogs of phosphoenolpyruvate, lphospholactate, d-phospholactate, and phosphoglycolate are potent competitive inhibitors. The inhibitor constant ( Ki) of l-phospholactate (2 μm) is 49-fold lower with Mn 2+ as the activator than with Mg 2+. An analysis of the competitive inhibition by portions of the l-phospholactate molecule (i.e., l-lactate, methyl phosphate, and phosphite) indicates this 49-fold lowering is due to increased interaction of the phosphoryl group and, to a lesser extent, of the carboxyl and C-O-P bridge oxygen of l-phospholactate with the enzyme metal complex. The results provide indirect evidence for phosphoryl coordination by the enzyme-bound divalent cation. 相似文献
9.
Barley, Panicum milioides and Panicum maximum were exposed to 14CO 2 near their photosynthetic CO 2 compensation points and their respective 14C-products were determined. In short exposure times Panicum maximum had 100% of its 14C in malate and aspartate whereas Panicum milioides and barley had 16 and 3% of their respective 14C in C 4 organic acids. Near the respective CO 2 compensation points a linear relationship occurs in plotting the ratio of glycine, serine, and glycerate to C 4 organic acids. The ratio of ribulose 1,5-bisphosphate oxygenase to phosphoenolpyruvate carboxylase is linear with their CO 2 compensation points. The photosynthetic CO 2 compensation point apparently is controlled by the activity of enzymes producing photorespiration metabolites and the activity of phospheonolpyruvate carboxylase. 相似文献
10.
The photosynthetic carbon fixation pathways and levels of carbon-fixing enzymes of four dominant submersed macrophytes of Lawrence Lake, southern Michigan, were investigated during the main growth season (May to November). All four species ( Scirpus subterminalis Torr., Najas flexilis (Willd.) Rostk. and Schmidt, Potamogeton praelongus Wulf., and Myriophyllum heterophyllum Michx.) were C 3 plants based on their patterns of 14C pulse-chase incorporation. High levels of phosphoenolpyruvate carboxylase were also found in these species. These levels, as well as the ribulose 1,5-biphosphate carboxylase/phosphoenolpyruvate carboxylase ratio of the leaves, varied throughout the growing season and exhibited highest values in July. No shift in carbon fixation pathways, however, could be detected from July to October. The possible functions of phosphoenolypyruvate carboxylase in these plants, as well as the significance of C 3 metabolism in submersed plants of temperate lakes, are delineated. 相似文献
11.
Ulva, a common green seaweed, performs at the biochemical level as a typical C 3 plant. Over 90% of label was found in glycerate 3-phosphate following a 3 second 14C pulse in the light, and the label was subsequently transferred to sugars. Also, the level of ribulose-1,5-bisphosphate carboxylase activity in crude extracts was about 10 times higher than that of phosphoenolpyruvate carboxylase. Concerning gas exchange, photosynthetic rates of Ulva showed no O 2 sensitivity, indicating that photorespiratory CO 2 losses are repressed as in C 4 plants. This apparent anomaly could be explained by the efficient HCO 3− uptake system of Ulva which might concentrate CO 2 to the chloroplasts, thus suppressing the oxygenase activity of ribulose-1,5-bisphosphate carboxylase. 相似文献
12.
The effects of Cu 2+, Zn 2+, Cd 2+ and Pb 2+ on growth and the biochemical characteristics of photosynthesis were more expressed in barley ( Hordeum vulgare L.) than in maize ( Zea mays L.) seedlings. The barley and maize seedlings exhibited retardation in shoot and root growth after exposure of Cu 2+, Cd 2+ and Pb 2+. The Zn 2+ions practically did not influence these characteristics. The total protein content of barley and maize roots declined with an increase in heavy metal ion concentrations. The protein content of barley shoots was only slighly decreased with an increase in heavy metal ion concentrations, but the protein content in maize shoots was increased under the same conditions. The chlorophyll content was decreased in barley shoots and increased in maize. The ribulose-l,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activities were decreased drastically by Cu 2+, Cd 2+ and Pb 2+ in the in vivo experiments. The tested heavy metal ions affect photosynthesis probably mainly by inhibition of these key carboxylating enzymes: this mechanism was studied in the in vitro experiments. 相似文献
13.
Wheat seedlings ( Triticum vulgare) treated with 1 m m KNO 3 or NaNO 3, in the presence of 0.2 m m CaSO 4, were compared during a 48-hour period with respect to nitrate uptake, translocation, accumulation and reduction; cation uptake and accumulation; and malate accumulation. Seedlings treated with KNO 3 absorbed and accumulated more nitrate, had higher nitrate reductase levels in leaves but less in roots, accumulated 17 times more malate in leaves, and accumulated more of the accompanying cation than seedlings treated with NaNO 3. Within seedlings of each treatment, changes in nitrate reductase activity and malate accumulation were parallel in leaves and in roots. Despite the great difference in malate accumulation, leaves of the KNO 3-treated seedlings had only slightly greater levels of phosphoenolpyruvate carboxylase than leaves of NaNO 3-treated seedlings. NADP-malic enzyme levels increased only slightly in leaves and roots of both KNO 3- and NaNO 3-treated seedlings. The effects of K + and Na + on all of these parameters can best be explained by their effects on nitrate translocation, which in turn affects the other parameters. In a separate experiment, we confirmed that phosphoenolpyruvate carboxylase activity increased about 2-fold during 36 hours of KNO 3 treatment, and increased only slightly in the KCl control. 相似文献
14.
Summary This communication reports the photoautotrophic growth of hormone and vitamin independent cell suspension cultures of Chenopodium rubrum. The transfer of cells from stationary growth into fresh culture medium results in a high protein formation, followed by an exponential phase of cell division, whereas the onset of rapid chlorophyll formation is delayed for 4 days. At the stage of most rapid cell division there is no net synthesis of starch and sugar. When the cells enter stationary growth, there is a progressive accumulation of chlorophyll, sugar, and starch.Photoautotrophic cell cultures assimilate about 80–90 mol CO 2/mg chlorophyll X hour. Dark CO 2 fixation is about 3.7% to 2.2% of the light values during exponential and stationary growth, respectively. As shown by short-term 14CO 2 fixation, CO 2 is predominantly assimilated through ribulosebisphosphate carboxylase via the Calvin pathway. There is a significant increase in the 14C label of C 4 carboxylic acids in exponentially dividing cells as compared to cells from stationary growth. The in vitro activity of phosphoenolpyruvate carboxylase and ribulosebisphosphate carboxylase is almost equal during exponential cell division. A decrease in cell division activity is accompanied by a significant change in the specific activities of both carboxylation enzymes. In non dividing cells from stationary growth the activity of ribulosebisphosphate carboxylase is greately enhanced and that of phosphoenolpyruvate carboxylase is reduced, documenting the development of carboxylation capacities typical for C 3-plants.The experimental results provide evidence that phosphoenolpyruvate carboxylase activity might be regulated by ammonia and could be involved in anaplerotic CO 2 fixation which supplies carbon skeletons of the citric acid cycle.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- EDTA
ethylene-diamine-tetraacetic acid
- FDP
fructose bisphosphate
- F-6-P
fructose-6-phosphate
- G-6-P
glucose-6-phosphate
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- PGA
3-phosphoglyceric acid
- PEP
phosphoenolpyruvate
- RuDP
ribulosebisphosphate 相似文献
15.
Ribulose 1,5-bisphosphate carboxylase/oxygenase purified from malate-grown Thiocapsa roseopersicina required Mg 2+ for the activation of both carboxylase and oxygenase activities. Mg 2+ was either not required or required at very low concentrations for catalysis by both enzyme activities. EDTA and dithiothreitol had no effect on ribulose 1,5-biphosphate oxygenase. The K0.5 values with respect to Mg 2+ for activation of the carboxylase and oxygenase activities were 8.4 and 2 mm, respectively. Ribulose 1,5-biphosphate carboxylase and oxygenase activities revealed differential sensitivities to 6-phosphogluconate. This ligand at 1 mm inhibited the carboxylase activity 30%, whereas the oxygenase activity was inhibited by 69%. 相似文献
16.
Illumination (22,000 lumens per meter 2) of etiolated maize plants for 80 hours brings about a 5-fold increase in phosphoenolpyruvate carboxylase activity per unit of protein. An increase in carboxylase protein and incorporation of [ 35S]methionine into the protein occurs simultaneously with the activity increase. In green plants, the level of phosphoenolpyruvate carboxylase protein and enzyme activity is dependent on the intensity of light during growth. These results are consistent with the conclusion that the activity increase results from light-stimulated de novo synthesis of phosphoenolypyruvate carboxylase protein. 相似文献
17.
Ribulose-1,5-bisphosphate caryboxylase-oxygenase is activated by CO 2 and Mg 2+ in a process distinct from catalysis. The effect of chloroplast metabolites as they separately influenced either activation or catalysis of tobacco carboxylase was examined. Of the 28 metabolites examined, 13 effected activation of the carboxylase. The strongest positive effectors were NADPH, gluconate-6- P, glycerate-2- P, and glycerate-3- P. Negative effectors included ribose-5- P, fructose-6- P, glucose-6- P, and pyrophosphate. The concentration of CO 2 or Mg 2+ necessary to produce half-maximal activation is defined as Kact. NADPH and gluconate-6- P decreased the Kact(CO 2) from 43 to 7.4 and 3.5 μm, respectively (pH 8.0, 5 mm MgCl 2). They also decreased the Kact(M.g 2+), but had little affect on the affinity of the enzyme for CO 2 during the catalytic process. Increasing Mg 2+ concentration decreased the Kact(CO 2) and increasing CO 2 concentration decreased the Kact-(Mg 2+). NADP + and gluconate-6- P also affected the pH profile of activation, shifting it toward lower pH values. Changes in activation had no effect on the pH profile for catalysis of CO 2 fixation. Effectors influenced ribulose-1,5-bisphosphate oxygenase in a manner analogous to the carboxylase. At air levels of O 2 and CO 2, the ratio of carboxylase to oxygenase activity was not changed by the presence of effectors, including hydroxylamine. 相似文献
18.
The carbon dioxide compensation concentration of Panicum milioides was less than that of soybean over the range of 15 to 35 C. In soybean ( Glycine max [L.] Merr. cv. Wayne), the compensation concentration was directly proportional to O 2 concentration. In P. milioides, the compensation concentration was near zero up to 10% O 2 and then increased linearly with higher O 2, although the slope of the response was less than that in soybean. Leaf extracts of P. milioides contained 3-fold higher phosphoenolpyruvate carboxylase activity than soybean leaf extracts. Oxygen inhibition of photosynthesis and carboxy-lation efficiency was less in P. milioides than that observed in soybean. The affinity of P. millioides ribulose-1,5-di-P carboxylase for CO 2 appeared to be slightly greater than that of soybean. The affinity of both enzymes for O 2 was similar. The reduced response of the compensation concentration and photosynthesis to O 2 in P. milioides may be explained by photosynthetic phosphoenolpyruvate carboxylase fixation and by an apparent increased affinity of ribulose-1,5-di-P carboxylase for CO 2. 相似文献
19.
Young bean plants ( Phaseolus vulgaris L. var Saxa) were fed with 3.5 or 10 millimolar N in either the form of NO 3− or NH 4+, after being grown on N-free nutrient solution for 8 days. The pH of the nutrient solutions was either 6 or 4. The cell sap pH and the extractable activities of phosphoenolpyruvate carboxylase and of pyruvate kinase from roots and primary leaves were measured over several days. The extractable activity of phosphoenolpyruvate carboxylase (based on soluble protein) from primary leaves increased with NO3− nutrition, whereas with NH4+ nutrition and on N-free nutrient solution the activity remained at a low level. Phosphoenopyruvate carboxylase activity from the roots of NH4+-fed plants at pH 4 was finally somewhat higher than from the roots of plants grown on NO3− at the same pH. There was no difference in activity from the root between the N treatments when pH in the nutrient solutions was 6. The extractable activity of pyruvate kinase from roots and primary leaves seemed not to be influenced by the N nutrition of the plants. The results are discussed in relation to the physiological function of both enzymes with special regard to the postulated functions of phosphoenolpyruvate carboxylase in C3 plants as an anaplerotic enzyme and as part of a cellular pH stat. 相似文献
20.
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 CO 2/1) and low (6 to 8 μl CO 2/l) CO 2 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 C 3 and C 4 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 (HCO 3 ?) 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 (Mg 2+) 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 C 4 acid inhibition and protection were not reliable methods to distinguish C 3 and C 4 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 C 3 and C 4 species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C 4 type. 相似文献
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