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1.
Immunological characterization of in vitro forms of homoserine dehydrogenase from carrot suspension cultures 总被引:3,自引:2,他引:1
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Multiple forms of homoserine dehydrogenase (HSDH) from carrot (Daucus carota L.) have been identified. One form of HSDH (T-form) has a relative molecular weight of 240,000 and is strongly inhibited by threonine. Another form (K-form) has a relative molecular weight of 180,000 and is insensitive to inhibition by threonine. The interconversion of these two forms is dependent upon the presence or absence of threonine and potassium. Polyacrylamide electrophoretic gels stained for HSDH activity and protein, paralleled with Western blot analysis, verified the interconversion of the T- and K-forms in 5 millimolar threonine and 100 millimolar potassium, respectively. Carrot HSDH also aggregates to form higher molecular weight complexes of 240,000 up to 720,000 Mr. Polyclonal antibody from mouse was raised against the T-form (240,000 Mr) of carrot HSDH. Specificity of the mouse antisera to carrot HSDH was verified by immunoprecipitation and Western blot analysis. The T-form, K-form, and all of the higher molecular aggregates of carrot HSDH cross-reacted with the anti-HSDH antiserum. The antiserum also cross-reacted with soybean HSDH, but did not cross-react with either of the two HSDH forms found in Escherichia coli. A model for the in vivo regulation of threonine biosynthesis in the chloroplast is presented. The model is based on the interconversion of the HSDH forms by potassium and threonine. 相似文献
2.
Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex : Does Photorespiratory Ammonium Influence Mitochondrial Carbon Metabolism? 总被引:4,自引:2,他引:2
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Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na+. NH4+, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K+ in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na+ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH4+ is discussed in relation to a possible regulatory interaction between photorespiratory NH4+ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex. 相似文献
3.
Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts 总被引:14,自引:9,他引:5
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Mills WR 《Plant physiology》1980,65(6):1166-1172
The metabolism of 14C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [14C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-14C]diaminopimelic acid was readily converted into [14C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO2 fixation. 相似文献
4.
Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties 总被引:3,自引:2,他引:1
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Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP+ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP+. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.
Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, Km of NADP+ being 2.5-fold higher for pI 4.3 form. Free ATP (Km = 0.64 millimolar) and ADP (Km = 1.13 millimolar) act as competitive inhibitors with respect to NADP+ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.
相似文献5.
Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg2+ or Mn2+, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and Ki for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine. 相似文献
6.
Aspartate kinase and homoserine dehydrogenase activity were assayed in a dialyzed cell-free extract ofCandida utilis. Aspartate kinase was partly inhibited by ATP-Mg and by Mg2+ alone. There appear to be two isoenzymes of aspartate kinase in the yeast, one heatlabile, the other relatively heat-stable.
The first is subject to feedback inhibition by threonine, the other is threonine-resistant. Neither aspartate kinase nor homoserine
dehydrogenase is the rate-limiting enzyme in methionine biosynthesis. Homoserine dehydrogenase measured in the forward direction
showed an activity five times higher than aspartate kinase. No regulatory interaction could be demonstrated for this enzyme.
No repression of aspartate kinase and homoserine dehydrogenase synthesis by threonine, methionine or both amino acids was
observed. 相似文献
7.
Homoserine dehydrogenase (HSD) was partially purified from castor bean, pea and wheat seedlings. The enzyme from pea had a MW of 75 000 and no sensitivity to threonine when measured in the direction of homoserine formation (forward reaction). The enzyme purified from castor bean had a MW of 290 000–350 000 and exhibited an almost complete inhibition by 1 mM threonine. Furthermore, this enzyme exhibited a polymeric nature as shown by polyacrylamide electrophoresis of the desensitized preparation and by SDS electrophoresis of the native enzyme. In wheat two isoenzymes were separated by gel filtration on Sephadex G 200. The fast-moving fraction (HSD I) was completely inhibited by threonine and exhibited a MW of 280 000, while the slow-moving fraction (HSD II) was insensitive to threonine and had a MW of 75 000. The sensitive enzyme from wheat and castor bean showed an almost absolute requirement for K+. The enzyme from pea and the insensitive form from wheat did not show a requirement for K+. For the wheat enzyme the effect of several amino acids and the main kinetic constants were studied. 相似文献
8.
Arginase (EC 3.5.3.1) was purified to homogeneity from cytosol of soybean, Glycine max, axes by chromatographic separations on Sephadex G-200, DEAE-sephacel, hydroxyapatite, and arginine-affinity columns. The molecular weight of the enzyme estimated by pore gradient gel electrophoresis was 240,000, while sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a single band at the molecular weight of 60,000. The optimal pH for activity was 9.5 and the Km value was 83 millimolar. The enzyme was stimulated by polyamines such as putrescine. 相似文献
9.
Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction 总被引:19,自引:18,他引:1
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The (K+,Mg2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg2+, the enzyme was further activated by monovalent cations (K+, NH4+, Rb+ Na+, Cs+, Li+). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K+ approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K+, and preferred Mn2+ to Mg2+. The results demonstrate that the (K+,Mg2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots. 相似文献
10.
Distribution and Properties of a Potassium-dependent Asparaginase Isolated from Developing Seeds of Pisum sativum and Other Plants 总被引:11,自引:6,他引:5
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Sodek L 《Plant physiology》1980,65(1):22-26
Asparaginase (EC 3.5.1.1) was isolated from the developing seed of Pisum sativum. The enzyme is dependent upon the presence of K+ for activity, although Na+ and Rb+ may substitute to a lesser extent. Maximum activity was obtained at K+ concentrations above 20 millimolar. Potassium ions protected the enzyme against heat denaturation. The enzyme has a molecular weight of 68,300. 相似文献
11.
All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD+ and NADP+. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH4Cl and 1.5 at 12.5 millimolar NH4Cl. Km values for NH4+ and NAD(P)H are reduced at low salt concentrations. The low Km values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations. 相似文献
12.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1988,952(2):222-229
Homogeneous preparations of l-threonine dehydrogenase (l-threonine: NAD+ oxidoreductase, EC 1.1.1.103) from Escherichia coli K-12, after having been dialyzed against buffers containing Chelex-100 resin, have a basal level of activity of 10–20 units/mg. Added Cd2+ stimulates dehydrogenase activity approx. 10-fold; this activation is concentration-dependent and is saturable with an activation Kd = 0.9 μM. Full activation by Cd2+ is obtained in the absence of added thiols. The pH-activity profile of the Cd2+-activated enzyme conforms to a theoretical curve for one-proton ionization with a pKa = 7.85. Mn2+, the only other activating metal ion, competes with Cd2+ for the same binding site. Km values forl-threonine and NAD+ as well as the Vmax for ‘demetallized’, Cd2+-activated, and Mn2+-activated threonine dehydrogenase were determined and compared. 相似文献
13.
Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata) 总被引:15,自引:7,他引:8
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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.
The activity of malic enzyme fromEscherichia coli was unaffected by the monovalent cations Na+ or Li+ at 10 mM. At 100 mM, Li+ or Na+ inhibited the enzyme activity by 88% and 83%, respectively. However, the enzyme activity was stimulated by 40–80-fold with 10 mM K+, Rb+, Cs+, or NH 4 + . Less stimulation was observed with 100 mM of these stimulating cations. The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol. The regenerated enzyme was inactivated by 5,5′-dithiobis(2-nitrobenzoic acid). The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by eithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN. The thiocyanoylated enzyme was insensitive to K+ stimulation, which suggested the essentiality of the sulfhydryl groups of theE. coli malic enzyme. 相似文献
15.
Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves 总被引:1,自引:1,他引:0
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Randall DD 《Plant physiology》1981,68(1):70-73
The subcellular location of NADP+-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP+-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP+-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP+-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent Km values of 11 micromolar (NADP+), 35 micromolar (isocitrate), 78 micromolar (Mn2+), 0.3 millimolar (Mg2+) and showed optimum activity at pH 8 to 8.5 with Mn2+ and 8.8 to 9.2 with Mg2+. The NADP+-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP+-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action. 相似文献
16.
Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction
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Malate oxidation supported C2H2 reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O2 tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (Km = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (Km = 2.32 millimolar). Both enzymes exclusively required NAD+ as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N2 fixation was demonstrated. The inhibition of O2 consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O2, is important in a general strategy of this legume to maintain N2 fixation under O2 limited conditions. 相似文献
17.
Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD+ Oxidoreductase 总被引:7,自引:1,他引:7
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Todd L. Talarico Lars T. Axelsson James Novotny Mitra Fiuzat Walter J. Dobrogosz 《Applied microbiology》1990,56(4):943-948
Lactobacillus reuteri utilizes exogenously added glycerol as a hydrogen acceptor during carbohydrate fermentations, resulting in higher growth rates and cell yields than those obtained during growth on carbohydrates alone. Glycerol is first converted to 3-hydroxypropionaldehyde by a coenzyme B12-dependent glycerol dehydratase and then reduced to 1,3-propanediol by an NAD+ -dependent oxidoreductase. The latter enzyme was purified and determined to have a molecular weight of 180,000; it is predicted to exist as a tetramer of identical 42,000-molecular-weight subunits. 相似文献
18.
Evidence in support of the hypothesis of gene expression and subunit association suggested earlier for Triticum alcohol dehydrogenase has been obtained through purification and partial characterization of the enzyme from tetraploid wheat. Three isozymes of alcohol dehydrogenase were separated and purified to apparent homogeneity using streptomycin sulfate precipitation, gel filtration chromatography, and anion exchange chromatography. The isozymes are dimers with the same molecular weight (116,000 ± 2,000), but significantly different isoelectric pH values. The Michaelis constants for NAD+ and ethanol are 0.1 millimolar and 12 millimolar, respectively. The substrate specificity of the three alcohol dehydrogenase isozymes was investigated. 相似文献
19.
Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana 总被引:1,自引:1,他引:0
The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD+ oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The Km values for α-ketoglutarate, NADH, NH4+, NAD+, and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the Km for α-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the Km values for NADH and NAD+ were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between α-ketoglutarate and an ε-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH4+-inducible glutamate dehydrogenase in this organism. 相似文献
20.
Summary
Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case ofEs-cherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD– or NADP+ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1mm methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, Km for substrate and cofactors), it resembles its counterpart inE. coli K12. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8m urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity ofS. marcescens unlike inSalmonella typhimurium andE. coli K12 where it is a minor or a negligible component. 相似文献