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1.
Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn2+ > Mn2+ > Mg2+. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn2+ > Zn2+ > Mg2+. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K+, then Rb+ and NH4+, whereas the enzyme had very little activity with Na+, Li+, or Cs+. Na+ did not interfere with the activation by K+. The activation of the kinase by K+, NH4+, and Rb+ followed Michaelis-Menten kinetics, and the apparent Km values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent Km value of the enzyme for pyridoxine and had little or no effect on the Km for ZnATP2? or the V of the kinase-catalyzed reaction.  相似文献   

2.
The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg2+, Co2+, Mn2+, Ni2+, and Zn2+ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg2+ and varying concentrations of Mn2+ or of Co2+. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co2+ and varying Mn2+. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn2+ is a better activator of the enzyme than is Mg2+, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni2+ or Zn2+ with Mg2+ suggests that Ni2+ and Zn2+ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg2+. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity.  相似文献   

3.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified to homogeneity with about 29% recovery from immature pods of chickpea using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sephadex G-200. The purified enzyme with molecular weight of about 200,000 daltons was a tetramer of four identical subunits and exhibited maximum activity at pH 8.1. Mg2+ ions were specifically required for the enzyme activity. The enzyme showed typical hyperbolic kinetics with phosphoenolpyruvate with a Km of 0.74 millimolar, whereas sigmoidal response was observed with increasing concentrations of HCO3 with S0.5 value as 7.6 millimolar. The enzyme was activated by inorganic phosphate and phosphate esters like glucose-6-phosphate, α-glycerophosphate, 3-phosphoglyceric acid, and fructose-1,6-bisphosphate, and inhibited by nucleotide triphosphates, organic acids, and divalent cations Ca2+ and Mn2+. Oxaloacetate and malate inhibited the enzyme noncompetitively. Glucose-6-phosphate reversed the inhibitory effects of oxaloacetate and malate.  相似文献   

4.
Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been partially purified by carboxymethylcellulose chromatography, and gel filtration. The enzyme is unstable in aqueous solution and requires the presence of a thiol protecting reagent as well as glycerol for the maintenance of activity. Dithiothreitol activates as well as stabilizes the enzyme. Phosphoenolpyruvate allosterically activates trypanosome pyruvate kinase whereas hyperbolic kinetics are found when ADP is the variable substrate. Mg2+ or Mn2+ ions and a monovalent cation are essential for enzyme activity. Fructose 1,6-diphosphate acts as a heterotropic allosteric activator, markedly decreasing the S0.5 value for phosphoenolpyruvate from 1.34 to 0.25 mm at 1 mm fructose 1,6-diphosphate and transforms the phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. The enzyme has a pH optimum of 6.5–7.0 and a molecular weight of 270,000 ± 27,000 as estimated by gel chromatography. Purine nucleotides are the preferred coenzymes for the reaction, having much lower Km values than the pyrimidine nucleotides. The possible role of pyruvate kinase in the regulation of glycolysis in T. brucei is discussed.  相似文献   

5.
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 (pI = 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 Mg2+ or Mn2+ 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 Mn2+ as the activator than with Mg2+. 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   

6.
Amino acid effector binding to rabbit muscle pyruvate kinase   总被引:1,自引:0,他引:1  
l-Phenylalanine, an allosteric inhibitor of rabbit muscle pyruvate kinase, is shown to bind to the tetrameric enzyme in a ratio of 4 moles effector per mole of tetramer. This binding is slightly cooperative in the absence of divalent cation activators, but the cooperativity is strongly increased when measured in the presence of 2.5 mm Mg2+ or Mn2+. The effector affinity is somewhat decreased under these conditions. l-Alanine was known to antagonize all measured phenylalanine effects and is shown here to also bind to 4 sites on the protein. The binding is noncooperative and little affected by the presence of the divalent activating cations. Competition experiments with phenylalanine and alanine suggest competition for the same site. Substrate kinetic measurements at P-enolpyruvate and Mg2+ concentrations under 100 μm show considerable inhibition of the enzyme at phenylalanine concentrations around 100 μm, near the serum levels of the free amino acid. The approach to the phenylalanine-inhibited velocity occurs with half-times less than 1 sec.  相似文献   

7.
The kinetic properties of partially purified kidney cortex, liver and muscle isozymes of rat pyruvate kinase (EC 2.7.1.40) were compared. The liver and kidney cortex enzymes were isolated in forms which were homotropically activated by phosphoenolpyruvate and heterotropically activated by fructose-1,6-diphosphate. In the absence of added modulators, the liver enzyme was less active, but both isozymes were fruther inactivated by l-alanine, l-phenylalanine or ATP. The liver enzyme was relatively more sensitive to ATP, but less sensitive to l-phenylalanine. The muscle enzyme, on the other hand, was isolated in a more active form which was insensitive to ATP or l-alanine inhibition and of intermediate sensitivity to l-phenylalanine inhibition. In the presence of l-phenylalanine, muscle enzyme also underwent homotropic and heterotropic activation. Not any of the isozymes were inhibited by NADH.All three isozymes were activated by K+ or NH4+. NH4+ was the more effective activator for the kidney cortex or liver enzymes, in the former case because of a greater affinity, the latter because of a higher catalytic efficiency. Of the divalent cations tested only Mg2+ and Mn2+ activated. All three isozymes had lower maximal rates when activated by Mn2+, but this ion also consistently acted as a typical K-type activator.Evidence also was obtained which suggested that the change from one conformational form to another might take minutes and therefore, measured kinetic parameters could reflect conformational as well as catalytic phenomena. This observation, plus suggested independent subunit interactions, were considered to be evidence favoring a sequential rather than a concerted mechanism of conformational transition.  相似文献   

8.
Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl2, effectively separates the Mg2+-ATPase from (Na+ + K+)-ATPase present in the same tissue and yields the Mg2+-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg2+, Mn2+, Ca2+, Co2+, Fe2+, Zn2+, Eu3+, Gd3+, and VO2+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with Rf values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-32P]ATP, suggesting that this peptide may contain the active site of the Mg2+-ATPase. The Mg2+-ATPase activity is unaffected by the specific (Na+ + K+)-ATPase inhibitor ouabain.  相似文献   

9.
F. hepatica pyruvate kinase and phosphoenolpyruvate (PEP) carboxykinase were found to have properties of regulatory enzymes in the dissimilation of PEP and the control of metabolic flow. Mn2+ and K+ were required for pyruvate kinase activity. In the presence of fructose-1, 6-diphosphate (FDP), Mg2+ could substitute for Mn2+. FDP caused a 4-fold increase in the Mn2+ activated pyruvate kinase activity. This was accompanied by a 12-fold decrease in apparent Km(PEP) and a 3-fold decrease in apparent Km (ADP). ATP markedly inhibited F. hepatica pyruvate kinase, but this inhibition was relieved by FDP. Estimates of metabolic levels indicated that the pyruvate kinase is saturated with PEP and ADP in vivo, but will be highly sensitive to fluctuations in the physiological concentrations of FDP and ATP. NADH doubled the activity of the PEP carboxykinase reaction and decreased the apparent Km (PEP) for this enzyme 3-fold. While the maximal activity of the PEP carboxykinase reaction was substantially higher than the pyruvate kinase reaction, the steady state concentration of PEP suggests that the PEP carboxykinase will not be saturated with this substrate.  相似文献   

10.
Most C4 species are chilling sensitive and certain enzymes like pyruvate,Pi dikinase of the C4 pathway are also cold labile. The ability of cations and compatible solutes to protect maize (Zea mays) dikinase against cold lability was examined. The enzyme in desalted extracts at pH 8 from preilluminated leaves could be protected against cold lability (at 0°C) by the divalent cations Mn2+, Mg2+, and Ca2+. There was substantial protection by sulfate based salts but little protection by chloride based salts of potassium or ammonium (concentration 250 millimolar). The degree of protection against cold lability under limiting MgCl2 (5 millimolar) was pH sensitive (maximum protection at pH 8), but independent of ionic strength (up to 250 millimolar by addition of KCl). In catalysis Mg2+ is required and Mn2+ could not substitute as a cofactor. Several compatible solutes reduced or prevented the cold inactivation of dikinase (in desalted extracts and the partially purified enzyme), including glycerol, proline, glycinebetaine and trimethylamine-N-oxide (TMAO). TMAO and Mg2+ had an additive effect in protecting dikinase against cold inactivation. TMAO could largely substitute for the divalent cation and addition of TMAO during cold treatment prevented further inactivation. Cold inactivation was partially reversed by incubation at room temperature; with addition of TMAO reversal was complete. The temperature dependence of inactivation at pH 8 and 3 millimolar MgCl2 was evaluated by incubation at 2 to 17°C for 45 minutes, followed by assay at room temperature. At preincubation temperatures below 11°C there was a progressive inactivation which could be prevented by TMAO (450 millimolar). The results are discussed relative to possible effects of the solutes on the quaternary structure of this enzyme, which is known to dissociate at low temperatures.  相似文献   

11.
Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg2+ or Mn2+ indicate a biphasic response to divalent cations. Apparent Km values of 23 μm for free Mg2+ and 3.3 μm for free Mn2+ are obtained at low levels of added metal, while Km values of 0.50 mm for free Mg2+ and 0.43 mm for free Mn2+ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg2+-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent Km values for free ATP of 6.0 and 1.4 μm with Mg2+ and Mn2+, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that Km values for free ATP of 8.0 and 2.0 mm are obtained for Mg2+ and Mn2+, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg2+-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with Ki values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn2+ per 469,000 g of protein. The Mn2+ binding studies yield a KD for Mn2+ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn2+ at low Mn2+ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn2+ per single high affinity Mn2+ site. The KD for Mn2+ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn2+ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn2+ in the presence of the enzyme is also consistent with the tight binding of a single Mn2+ ion per 469,000 Mr protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn2+ at the single tight site, ?b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn2+ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg2+-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg2+-ATPase described here are discussed.  相似文献   

12.
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 Km 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.  相似文献   

13.
A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the Km of PEP was 0.14 millimolar and the Vmax was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg2+ ions (Km = 0.12 millimolar). Other divalent metal ions inhibited the Mg2+ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites.  相似文献   

14.
Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K+ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg+2 is optimal for the physiological reaction, however, Co+2, Mn+2, and Ni+2 also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.  相似文献   

15.
Reactivation of the pea mitochondrial pyruvate dehydrogenase complex was the result of dephosphorylation catalyzed by phospho-pyruvate dehydrogenase-phosphatase, an intrinsic component of the complex. Phosphatase activity was dependent upon divalent metal ions, with Mg2+ more effective than Mn2+ or Co2+. The Michaelis constants for Mg2+, Mn2+, and Co2+ were 3.8, 1.7, and 1.4 millimolar, respectively. Neither the rate nor the extent of activation of the phosphatase by Mg2+ or Mn2+ was effected by up to 100 units per assay of megamodulin. Calcium ions did not activate pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase, and low concentrations of Ca2+ antagonized activation by other divalent cations. Phosphatase activity was inhibited by fluoride and ortho-phosphate but not by molybdate or vanadate. Krebs cycle intermediates, adenylates, polyamines, amino acids, and phosphoamino acids were without effect upon pea mitochondrial phospho-pyruvate dehydrogenase-phosphatase activity in vitro.  相似文献   

16.
Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn2+ and Mg2+ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg2+ or Zn2+ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn2+ > Co2+ > Mg2+ > Mn2+ > Ca2+ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn2+ produces the same K m, for all the complexes and Mg2+ stimulates with a different K m. The results suggest that both Mg2+ and Zn2+ activate the membrane-bound pyrophosphatase but through different mechanisms.  相似文献   

17.
Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris   总被引:1,自引:1,他引:0  
ADP-glucose pyrophosphorylase was partially purified from Chlorella vulgaris 11h. 3-Phosphoglycerate activated the enzyme by lowering the Michaelis constant for glucose-1-phosphate (from 0.97 to 0.36 millimolar in the presence of 2 millimolar phosphoglycerate) and ATP (from 0.23 to 0.10 millimolar), as well as increasing the Vmax. Saturation curves for 3-phosphoglycerate were hyperbolic and the activator concentration at half Vmax value for 3-phosphoglycerate was 0.41 millimolar either in the presence or absence of phosphate. Phosphate inhibited the enzyme in a competitive manner with respect to glucose-1-phosphate, but did not affect the Michaelis constant value for ATP. 3-Phosphoglycerate changed neither the inhibitor concentration at half Vmax value of 1.0 millimolar for phosphate nor the hyperbolic inhibition kinetics for phosphate. The enzyme required divalent cations for its activity. The activation curves for Mn2+ and Mg2+ were highly sigmoidal. The activator concentration at half Vmax values for Mn2+ and Mg2+ were 2.8 and 3.7 millimolar, respectively. With optimal cations, the Michaelis constant values for ATP-Mn and ATP-Mg were 0.1 and 0.4 millimolar, respectively.  相似文献   

18.
The properties and role of the enzyme phosphoglycolate phosphatase in the cyanobacterium Coccochloris peniocystis have been investigated. Phosphoglycolate phosphatase was purified 92-fold and had a native molecular mass of approximately 56 kilodaltons. The enzyme demonstrated a broad pH optimum of pH 5.0 to 7.5 and showed a relatively low apparent affinity for substrate (Km = 222 micromolar) when compared to that from higher plants. The enzyme required both an anion and divalent cation for activity. Mn2+ and Mg2+ were effective divalent cations while Cl was the most effective anion tested. The enzyme was specific for phosphoglycolate and did not show any activity toward a variety of organic phosphate esters. Growth of the cells on high CO2 and transfer to air did not result in any significant change in phosphoglycolate phosphatase activity. Competitive inhibition of C. peniocystis triose phosphate isomerase by phosphoglycolate was demonstrated (Ki = 12.9 micromolar). These results indicate the presence of a specific noninducible phosphoglycolate phosphatase whose sole function may be to hydrolyze phosphoglycolate and prevent phosphoglycolate inhibition of triose phosphate isomerase.  相似文献   

19.
The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn2+ ions for activity; Mg2+ ions reduce the Km for Mn2+ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (kcat=2.7 s?1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.  相似文献   

20.
Pyruvate kinase was extracted from Me2CO-dried tissue of various parts of tomato plants. Recovery of the enzyme was improved by the inclusion of thiols in the extraction medium, and its stability was increased considerably in the presence of glycerol and to a lesser extent tetramethylammonium chloride. A phosphatase was present in the tissue extracts which hydrolyses phosphoenolpyruvate in the absence of added ADP. ATP inhibited pyruvate kinase but stimulated the phosphatase, while Mg2+ stimulated both enzymes. Data obtained suggest that tomato leaf pyruvate kinase has an absolute dependence on monovalent cations for activity, K+ being the principal activator. The phosphatase was inhibited non-selectively by monovalent cations. The total activity of pyruvate kinase and its concentration on a tissue fresh weight basis was greatest in the leaves, activity increasing with the maturity of the tissue. Less enzyme was present in roots, and least in the fruit.  相似文献   

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