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1.
Abstract. In the preliminary purification of Capsicum leaf nitrate reductase (EC 1.6.6.1), treatment of the crude extract on Sephadex G-25 was necessary to prevent a gelling of the extract and sedimentation of the enzyme. Its Km values for NADH and nitrate were estimated to be 9.3 and 105mmol m−3 ADP and ATP gave hyperbolic competitive inhibition, with respect to NADH, while the inhibition by AMP was linear competitive. Ki values calculated were: ADP and ATP approximately lmol m−3 and AMP 2.3 mol m−3 . Inhibition by ADP was not altered by reduced glutathione.
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity. 相似文献
The Capsicum nitrate reduclase was very susceptible to inhibition by NADH (in the absence of nitrate) and an in vivo assay showed that the activity of the enzyme was limited by the supply of nitrate. NADH and adenine nucleotide levels measured in the Capsicum leaf were used to estimate inhibition of nitrate reductase and a prediction was made of the nitrate reductase activity at different times in the photoperiod. This was shown to follow the same trend as the measured in vivo activity of the enzyme. Changes in adenine nucleotide levels had little effect on nitrate reductase activity. 相似文献
2.
Purification and characterization of adenine phosphoribosyltransferase from Arabidopsis thaliana 总被引:1,自引:1,他引:0
Adenine phosphoribosyltransferase (APRT; EC 2. 4,2. 7) from Arabidopsis thaliana was purified approximately 3800-fold, to apparent homogeneity. The purification procedure involved subjecting a leaf extract to heat denaturation, (NH4 )2 SO4 precipitation, Sephadex G-25 salt separation, ultracentrifugation and liquid chromatography on Diethylaminoethyl Sephacel, Phenyl Sepharose CL-4B, Blue Sepharose CL-6B and adenosine 5'-monophosphate-Agarose. The purified APRT was a homodimer of approximately 54 kDa and it had a specific activity of approximately 300 μmol (mg total protein)-1 min-1 . Under standard assay conditions, the temperature optimum for APRT activity was 65°C and the pH optimum was temperature dependent. High enzyme activity was dependent upon the presence of divalent cations (Mn2+ or Mg2+ ). In the presence of MnCl2+ other divalent cations (Mg2+ , Ca2+ , Ba2+ , Hg2+ and Cd2+ ) inhibited the APRT reaction. Kinetic studies indicated that 5-phosphoribose-1-pyrophosphate (PRPP) caused substrate inhibition whereas adenine did not. The Km for adenine was 4.5±1.5 μ M , the Km for PRPP was 0.29±0.06 m M and the Ki for PRPP was 1.96±0.45 m M . Assays using radiolabelled cytokinins showed that purified APRT can also catalyze the phosphoribosylation of isopentenyladenine and benzyladenine. The Km for benzyladenine was approximately 0.73±0.06 m M 相似文献
3.
Chloroplast glutathione reductase: Purification and properties 总被引:4,自引:0,他引:4
Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)−1 min−1 . The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The Km for oxidized glutathione was 11 μ M and the Km for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H2 O2 by reducing dehydroascorbate for ascorbate-linked reduction of H2 O2 . Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity. 相似文献
4.
The synthesis of homoglutathione (hGSH) by several plants of the tribe Phaseoleae is shown to be catalysed by a β-alanine-specific hGSH synthetase, Properties of the enzyme from Phaseolus coccineus L. cv. Preisgewinner were studied, using ammonium sulfate precipitates of primary leaf extracts. The hGSH synthetase showed a broad pH optimum at pH 8–9, an absolute requirement for Mg2+ , a stimulation by K+ , and a high affinity for γ-glutamylcysteine [Km (app.) 73 μ M ]. The enzyme exhibited a high specificity for β-alanine [Km (app.) 1.34 m M ] compared to glycine [Km (app.) 98 m M ]. Chloroplasts, isolated from the leaves of Phaseolus coccineus , contained about 17% of the hGSH synthetase activity in the leaf cells. 相似文献
5.
Diacetyl reductase from Kluyveromyces marxianus NRRL Y-1196 was purified 27.5-fold with a yield of 13% by ammonium sulphate fractionation, DEAE-anion exchange chromatography, hydroxyapatite chromatography and chromatofocusing. The purified enzyme was most active at pH 7.0 and exhibited optimal activity at 40°C. The K m and V max values for diacetyl were 2.5 mmol 1-1 and 0.026 mmol 1-1 min-1 , respectively. The enzyme did not react with monoaldehydes or monoketones, but reduced acetoin, diacetyl and methylglyoxal with NADH as a cofactor. The enzyme had an isoelectric point (pl) of pH 5.8, and its molecular weight was 50 kDa. 相似文献
6.
M. Belamri A. Hadj Sassi M. Savart A. Tantaoui-Elaraki P. Cottin 《Letters in applied microbiology》1994,19(6):410-413
A novel extracellular β-fructosidase produced by Bacillus stearothermophilus has been identified and purified. The purified enzyme, obtained by using successive QEAE Sepharose fast flow and Sephacryl S300 HR columns, has a 600 kDa relative molecular weight (Mr ) and is composed of 60 kDa subunits indicating a multimeric structure. The pH and temperature for optimal activity are 6.5 and 65°C respectively, the enzyme being thermostable at this temperature. The apparent Km values for sucrose and inulin are 3.56 mmol l-1 and 1 mmol l-1 respectively, the total invertase/total inulinase ratio being 4. 相似文献
7.
A procedure for the partial purification of a non-specific alkaline phosphatase (EC 3.1.3.1.) from the embryonic axes of chick-pea seeds is described. Ammonium sulphate precipitation, DEAE-cellulase chromatography, Sephacryl S-200 chroma-tography and polyacrylamide gel electrophoresis are the most important steps. The molecular weight of this non-specific enzyme, as determined by Sephacryl S–200 gel filtration and SDS–polyacrylamide gel electrophoresis, was estimated as being 68 and 78 kDa respectively; the optimum pH for p-nitrophenylphosphate hydrolysis was 7.5, and the Km for this artificial substrate was 0.5 mM. The enzyme catalyzes the hydrolysis of a variety of organic phosphate esters. The best substrates are: phos-phoenolpymvate (Km = 2.4 m M ), NADP+ (Km = 4.0 m M ), 5'-AMP (Km = 4.5 m M ), 5'-ADP (Km = 6.1 m M ) and ribose-5P (Km = 5.8 m M ); but it is unable to hydrolyze 5'-ATP, phosphocreatine and tripolyphosptiate. Phospate was a competitive inhibitor. Zn2+ , K+ , Hg2+ and Mo6+ were strong inhibitors, whereas F− and Ca2+ inhibited weakly; Co2+ and Ni2+ were activators. 相似文献
8.
Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups 总被引:2,自引:0,他引:2
Paulo Afonso Granjeiroa Carmen Veríssima Ferreirab José Mauro Granjeiroc Eulázio Mikio Tagac Hiroshi Aoyamaa 《Physiologia plantarum》1999,107(2):151-158
An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg−1 protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent Km value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu2+ and Zn2+ . The strong inhibition by p CMB, Cu2+ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPPi ) as substrate. The highest specificity constant (Vmax /Km ) was observed with KPPi , making it a potential physiological substrate. 相似文献
9.
Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K+ , Mg2+ -ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent Km (ATP) and Vmax of 0.75 m M and 79 μmol (mg protein)−1 h−1 , respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 m M and Vmax of 159 μmol (mg protein)−1 h−1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate. 相似文献
10.
Abstract NADP-dependent glutamate dehydrogenase (GDH; E.C.1.4.1.4) was purified from an obligate methylotroph Methylobacillus flagellatum using ammonium sulphate precipitation, DEAE-Sepharose and dye-ligand Procion red HE3B column chromatography and Sephacryl S-200 gel-filtration. The Mr of the native enzyme was estimated to be 300 000 (±5000). The enzyme consists of six identical subunits with an Mr of 47 000 (±3000) (SDS-PAGE). The enzyme has a pH optimum of 8.0 when participating in amination and 9.5 in deamination. Michaelis-Menten kinetics were observed for both reactions. The apparent Km values were 1.33 mM, 0.032 mM, 11.5 mM, 7.0 mM and 0.014 mM for α-ketoglutarate, NADPH, NH4 + , glutamate and NADP+ , respectively. The enzyme was highly specific for all the substrates and was insensitive to inhibitors. It plays an exclusively anabolic role in the cells. 相似文献
11.
Serine hydroxymethyltransferase from spinach leaf mitochondria. Purification and characterization 总被引:1,自引:0,他引:1
A mitochondrial serine hydroxymethyltransferase (EC 2.1.2.1) has for the first time been purified close to homogeneity from a photosynthetically active tissue, spinach ( Spinacea oleracea L. cv Viking II) leaves. The specific activity of the enzyme was 7.8 μmol (mg protein)−1 min−1 using L-serine as substrate. The enzyme was stable for at least 8 weeks at 4°C in the presence of folate. The pH optimum was at pH 8.5 where the enzyme had a Km for L-serine of 0.9 m M . Carboxymethoxylamine was a strong competitive inhibitor with a K1 of 1.4 μM. An absorption spectrum taken of the enzyme in the presence of glycine and tetrahydrofolate showed a peak at 492 nm, probably originating from a substrate-enzyme complex. The molecular weight obtained by gel filtration was 209 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified enzyme showed that the apparent molecular weight of the subunit was 53 kDa, indicating four subunits. 相似文献
12.
Amylase activity extracted from tulip ( Tulipa gesneriana L. cv. Apeldoorn) bulbs that had been stored for 6 weeks at 4°C was resolved to 3 peaks by anion-exchange chromatography on diethylaminoethyl-Sephacel. These 3 amylases exhibited different relative mobilities during non-denaturing polyacrylamide gel electrophoresis (PAGE). The most abundant amylase form (amylase I) was purified to apparent homogeneity using hydrophobic interaction chromatography, gel filtration and chromatofocusing. The apparent molecular mass of the purified amylase was estimated to be 51 kDa by sodium dodecyl sulfate-PAGE and 45 kDa by gel filtration chromatography. The purified amylase was determined to be an endoamylase (EC 3.2.1.1) based on substrate specificity and end-product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55°C. The apparent Km value with soluble starch (potato) was 1.28 mg ml−1 . The presence of Ca2+ increased the activity and thermal stability of the enzyme. The presence of dithiothreitol enhanced the activity, while β -mercaptoethanol and reduced glutathione had no significant effect. When pre-incubated in the absence of the substrate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) partially inhibited the enzyme. α -cyclodextrins or β -cyclodextrins had no effect on enzyme activity up to 10 m M . In addition to CaCl2 , CoCl2 slightly enhanced activity, while MgCl2 and MnCl2 had no significant effect at a concentration of 2 m M . ZnCl2 , CuSO4 , AgNO3 and EDTA partially inhibited enzyme activity, while AgNO3 and HgCl2 completely inhibited it at 2.0 m M . 相似文献
13.
Biochemical characterization of nitrate and nitrite reduction in the wild-type and a nitrate reductase mutant of soybean 总被引:1,自引:0,他引:1
Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild-type (cv. Williams) and mutant ( nr1 ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr1 soybean mutant (formerly LNR-2), had decreased NADH-NR, FMNH2 -NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH2 -NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH2 as a reductant source. Nitrite reductase activity was normal in the nr1 plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea-grown wild-type plants had 1) greater ability to use FMNH2 as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO3 − and HCO3 − , compared with inducible NR from NO3 − -grown nr1 plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity. 相似文献
14.
Abstract. Oxygen uptake characteristics of the roots of three Rumex species were compared, and related to kinetics of the respiratory system and to root anatomy. The observed differences could not be explained by differences in fundamental characteristics of the oxygen uptake system: with all three species, cytochrome-mediated respiration contributed 70% and cyanide-insensitive (alternative) respiration 30% of the total respiration rate, and apparent Km values of cytochrome oxidase were lower than those obtained for the alternative oxidase in all cases. However, differences in critical oxygen pressure for respiration (COPR) and in apparent Km for oxygen, were strongly correlated with differences in root porosity and root diameter. Km (O2 ) values at high and low temperatures were determined, and from Arrhenius plots of oxygen uptake rates between 11 and 32°C, the role of diffusional impedance could be estimated. Root respiration of Rumex maritimus and R. crispus , both with high root porosity, but differing in root diameter, had a low Km for oxygen (3–7 mmol m−3 ). In contrast with this were the responses of R. thvrsiflorus , which has thin roots but low root porosity: a high Km (10-20 mmol m−3 ) was found at all temperatures. The role of diffusional impedance as a function of temperature in oxygen uptake rate by the three species is discussed and related to the differential resistance of the species towards flooding. 相似文献
15.
16.
William J. Hunter 《Physiologia plantarum》1984,60(4):467-472
A nodule cytosol nitrite reductase was isolated from soybean [ Glyine max (L.) Mer. cv. Tracy] grown in the presence of nitrate. Enzyme activity increased when increased amounts of nitrate were supplied to the plant. A purification procedure involving ammonium sulfate precipitation, gel filtration, DEAE Sephadex and Blue Sepharose chromatography resulted in an activity capable of forming 6.7 μmol ammonia (mg protein)−1 min−1 . This represented a 235-fold increase in specific activity. The molecular weight, estimated by gel filtration, was 55 000. The pH optimum for activity was 7.1. Ammonia formed stoichiometrically as nitrice was consumed. From Lineweaver-Burk plots, Km values of 0.5m M for nitrite and 0.2m M for methyl viologen were calculated. Spectral data suggest the association of a heme chromophore with the enzyme. 相似文献
17.
NADP+ -isocitrate dehydrogenase (EC 1.1.1.42) was purified more than 1500-fold from the host-plant cytosol of Medicago sativa L. cv. Saranac root nodules by ion exchange and affinity chromatography. The forward reaction was characterized. The enzyme exhibited an absolute requirement for a divalent cation (preferably Mn2+ ), had a broad activity optimum from pH 7.5 to 9.0, and was most stable at pH 7.5. The apparent Arrhenius energy of activation was 70.7 kJ mol−1 (20 to 30°C) indicating that the reaction rate of the enzyme was relatively sensitive to temperature. The Km for isocitrate was 20 μ M and for NADP+ 10.7 μ M . Initial velocity and end product inhibition studies of the forward reaction indicate a random bi ter mechanism. End product studies indicated that NADPH was a competitive inhibitor and α-ketoglutarate was a non-competitive inhibitor with respect to isocitrate and NADP+ . Citrate was a competitive inhibitor with respect to isocitrate. Glutamine was identified as a positive effector when assays were conducted at non-saturating isocitrate concentrations. The potential significance of glutamine regulation of α-ketoglutarate production in a dinitrogen-fixing tissue is discussed. 相似文献
18.
Abstract Nitrate reduction to ammonia by marine Vibrio species was studied in batch and continuous culture. In pH-controlled batch cultures (pH 7.4; 50 mM glucose, 20 mM KNO3 ), the nitrate consumed accumulated to more than 90% as nitrite. Under these conditions, the nitrite reductase (NO− 2 → NH3 ) was severely repressed. In pH-controlled continuous cultures of V. alginolyticus with glucose or glycerol as substrates ( D = 0.045 h−1 ) and limiting N-source (nitrate or nitrite), nitrite reductase was significantly derepressed with cellular activities in the range of 0.7–1.2 μmol min−1 (mg protein)−1 . The enzyme was purified close to electrophoretic homogeneity with catalytic activity concentrations of about 1800 nkat/mg protein. It catalyzed the reduction of nitrite to ammonia with dithionite-reduced viologen dyes or flavins as electron donors, had an M r of about 50 000 (determined by gel filtration) and contained c-type heme groups (probably 4–6 per molecule). 相似文献
19.
Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)−1 min−1 ] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca2+ , and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent Km (SHAM) of ca 40 μ M (with NADH). The dependence of O2 consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H2 O2 was produced for every 1 mol O2 consumed. Endogenous catalase converted this H2 O2 to O2 and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O3 . SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed. 相似文献
20.
Leszek A. Kleczkowski Curtis V. Givan Douglas D. Randall Tadeusz Loboda 《Physiologia plantarum》1988,74(4):763-769
Leaf extracts from seven monocotyledonous and dicotyledonous species contained considerable levels of NADPH-dependent glyoxylate- and hydroxypyruvate reductase activities. These activities ranged from 0.02 to 0.22 μmol (mg protein)−1 min−1 . For all plants tested, the glyoxylate reductase (GR) activity, assayed with either NADPH or NADH, was sensitive to inhibition by acetohydroxamate, a glycine analogue. Hydroxypyruvate reductase (HPR) activities were unaffected by acetohydroxamate. Differential precipitation of soluble leaf proteins of spinach, pea and barley by ammonium sulfate (0–45% and 45–60% saturation) indicated the presence of at least three distinct reductases, which differed in their specificities for glyoxylate, hydroxypyruvate and NAD(P)H. For all species, the NADH-dependent HPR-activity was almost completely precipitated by low ammonium sulfate concentration (45%), while precipitation of the NADPH-GR, NADH-GR and, to some extent, NADPH-HPR activities required 60% ammonium sulfate. The NADPH-dependent GR and HPR activities had high affinity for glyoxylate and hydroxypyruvate, respectively, as indicated by low apparent Km values of 40–120 μ M . The occurrence of at least three distinct reductases utilizing hydroxypyruvate and/or glyoxylate as substrate was supported by antibody-precipitation studies using antibodies prepared against NADH(NADPH)-HPR, the well-known peroxisomal enzyme that also shows non-specific GR activity. These data are discussed with respect to recent reports on the purification and characterization of NADPH(NADH)-GR, and NADPH (NADH)-HPR, two cytosolic reductases, and the role is assessed for these enzymes in reducing hydroxypyruvate and glyoxylate that may be leaked from peroxisomes. 相似文献