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1.
The activity and decay characteristics of nitrate reductase from wheat (Triticum aestivum) were studied in crude, partially-purified and highly-purified preparations. The decay of nitrate reductase activity in crude extracts was due to spontaneous dissociation of the enzyme and to the effects of two decay factors, one present in the 0–30% and the other in the 50–70% saturated (NH4)2SO4 fraction of a crude extract. Low rates of factor-mediated NR decay in vitro were associated with high levels of NR activity in vivo.  相似文献   

2.
Soybean cell suspension cultures (Glycine max L. cv. Kanrich) grown on high-nitrogen medium produce 50 mU/g fresh wt of phenylalanine ammonia-lyase [EC 4.1.3.5] 7–9 days after inoculation. Nitrate was not limiting when the peak of enzyme activity was reached. Phenylalanine ammonia-lyase was purified 53-fold to essentially electrophoretic homogeneity from cell extracts with 10% recovery. The enzyme was stable in crude extracts and through most stages of purification. No activity could be detected with tyrosine as substrate in either crude extracts or purified enzyme. The electrophoretic mobility was somewhat less than that of the enzyme from maize but both eluted from an agarose column at the same position and the molecular weight of the subunit was similar for both enzymes. Thus the soybean enzyme is composed of four subunits and the native enzyme is ~330,000 Mr. The variation in structure and/or size and availability of hydrophobic regions among phenylalanine ammonia-lyases from four sources (potato, maize, Rhodotorula glutinis, and soybean) was shown by the different elution patterns they exhibited on columns of ω-aminoalkyl agarose (agarose-Cn-NH2, n = 0 to 8). The order of increasing hydrophobicity is soybean, potato, maize, R. glutinis. The soybean enzyme exhibited negative cooperativity before hydroxylapatite chromatography and positive cooperativity afterward. This is the first example of positive cooperativity observed for phenylalanine ammonia-lyase.  相似文献   

3.
A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg2+, Ag+, Zn2+, and KMnO4.  相似文献   

4.
Arima K  Yu J  Iwasaki S  Tamura G 《Applied microbiology》1968,16(11):1727-1733
A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg2+, Ag+, Zn2+, and KMnO4.  相似文献   

5.
Zinc-induced paracrystalline aggregation of glutamine synthetase   总被引:22,自引:0,他引:22  
The unique capacity of glutamine synthetase to form highly insoluble paracrystalline aggregates in the presence of Zn2+ and Mg2+ mixtures is the basis of a new simple procedure for the isolation of the enzyme from crude extracts of Escherichia coli. Under optimal conditions (pH 5.85, 25 °C, 1.5 mm ZnSO4 and 50 MgCl2 over 95% of the enzyme is precipitated from crude extracts; differential extraction of the precipitate with dilute buffer (pH 7.0) containing 2.5 mm MgCl2 leads to high yields of almost pure glutamine synthetase. Polyacrylamide gel electrophoresis of the purified enzyme shows it to consist of one major protein and two minor protein components, all of which exhibit glutamine synthetase activity. The major component appears to be identical with the enzyme previously isolated by the older more tedious procedure of Woolfolk et al. (1966). The γ-glutamyl transferase activity of enzyme isolated by the new procedure is the same as that isolated by the older method, but its biosynthetic activity is 25–35% lower. In all other respects examined (i.e., divalent ion specificity, pH optimum, apparent Km values for substrates, susceptibility to feedback inhibition and physical properties) enzymes prepared by the old and the new procedures are indistinguishable. From studies with pure glutamine synthetase isolated by either procedure, it has been established that paracrystalline aggregation does not occur until 9–10 equivs of Zn2+ are bound per mole of enzyme. The high specificity of Zn2+ in inducing enzyme aggregation, suggests that its binding provokes a unique conformational state of the enzyme. This is supported by the fact that addition of Zn2+ to relaxed (divalent cation free) enzyme elicits a change in the ultraviolet spectrum of the enzyme that is qualitatively different from that caused by either Mg2+ or Mn2+. Moreover, in contrast to Mg2+, the binding of Zn2+ decreases the fluorescence associated with the binding of 2-p-toludinyl-naphthalene-6-sulfonic acid to the enzyme, suggesting that Zn2+ binding is accompanied by a decrease in the number of exposed hydrophobic regions on the enzyme.  相似文献   

6.
Endoglucanase production was carried out using in-house isolate Aspergillus terreus on rice straw under solid state fermentation. An increase of 1.25-fold endoglucanase production was obtained under optimized conditions using response surface methodology. The enzyme was purified to homogeneity by gel filtration chromatography. Its molecular weight was determined as 28.18 kDa by gel filtration and 29.13 kDa on SDS-PAGE. The enzyme displayed maximum activity at 50 °C and pH 4.8. It was stable for 240 min at 50 °C and 120 min at 60 °C but rapidly inactivated at 70 °C. The purified enzyme was specific towards carboxymethyl-cellulose but showed no activity for cellobiose or xylan. Maximum velocity (Vmax) and KM were 16.15 μmol min−1 mg−1 and 12.01 mg ml−1, respectively. AgNO3, KCl, NaCl, and MnSO4 were found to inhibit enzyme activity while CaCl2 and ZnSO4 activated the enzyme. Internal peptide mass fingerprinting analysis identified that the protein belongs to GH12 superfamily endoglucanases. External supplementation of the purified enzyme to the crude cellulase showed 38.7% increase in saccharification efficiency of the delignified rice straw compared to the crude cellulase alone. The results demonstrated that the addition of GH 12 family purified endoglucanase to the crude cellulase can efficiently convert lignocellulosic biomass to fermentable sugars.  相似文献   

7.
A proteolytic enzyme capable of cleaving intact proteins and synthetic substrates α?N?benzoyl?DL?arginine β?naphthylamide (Bz-Arg-NNap), α-N-benzoyl-L-arginine p-nitroanilde (Bz-Arg-NPhNO2), and α-N-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) was purified 92– fold from the rabbit testes. The enzyme exhibited optimal activity at pH 9.0 and 50°C. The polyacrylamide gel electrophoresis and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme demonstrated multiple forms; the major band in the SDS-polyacrylamide gel electrophoresis corresponded to a Mt 48,000. The same value was established by the gel filtration over Sephadex G-75. The rabbit testicular alkaline proteinase (TAP) resembled acrosin in the hydrolysis of Bz-Arg-OEt. However, CaCl2, a potential stimulator of acrosin activity, inhibited the alkaline proteinase. The strong inhibitors of acrosin, eg pheny methyl sulphonyl fluoride (PMSF), tosyl lysine chloromethyl ketone (TLCK), and benzamidine did not inhibit the alkaline proteinase. TAP was activated by an acrosin inhibitor isolated from the rabbit testes. Since 0.5 M KCl was necessary for complete extraction of the enzyme and the bulk of the activity was present in 9,000g pellet of the testicular homogenate. The alkaline proteinase appeared to be associated with the membranous structures.  相似文献   

8.
《Phytochemistry》1987,26(12):3233-3234
A crude enzyme preparation obtained from cell suspension culture of Catharanthus roseus converted 3′,4′-anhydrovinblastine to vinblastine, an anticancer agent. NADH and MnCl2 in the reaction mixture enhanced vinblastine yields.  相似文献   

9.
A recombinant insecticide-resistant mosquito carboxylesterase B1 was purified to homogeneity from an Escherichia coli expression system. After non-denaturing electrophoresis, active carboxylesterase B1 bands were identified using fast blue RR. Lineweaver–Burk plots of the crude and purified CaE B1 indicate that this enzyme obeys Michaelis–Menten kinetics with Km value for malathion of 39.3 and 67.4 mM. The Vm of purified enzyme is approximately 17-folds of the value determined in crude homogenate. Carboxylesterase B1 detoxification of parathion had a major limitation which is the 1:1 stoichiometry. To improve the effectiveness of enzymatic detoxification, we developed an approach in which the catalytic activity of organophosphorus compound-inhibited carboxylesterase B1 was restored by having sufficient amounts diacetylmonoxime. It was demonstrated that repeated addition of 25 times the molar concentration of parathion to carboxylesterase B1 in the presence of 4 mM diacetylmonoxime every 2 h did not result in significant inhibition of the enzyme. Consequently the stoichiometry of enzyme detoxification is higher than 64: 1 for parathion.  相似文献   

10.
Phenylalanine transfer ribonucleic acid synthetase from Drosophila melanogaster has been purified 1400-fold over a crude 230,000g supernatant fraction. The optimum activity of the enzyme occurs at magnesium concentrations above 10 mm at 37 °C and pH 7.5. At a 50 mm Mg2+ concentration, NH4+ stimulates the ATP-PP1 exchange reaction as much as 2-fold. Ammonium chloride causes an increase in the V with no change in the Km with phenylalanine as substrate. Homologous (Drosophila) tRNA, in the presence of NH4+, further stimulates the ATP-PPi, exchange reaction but inhibits the reaction in the absence of NH4+.In the presence of its substrates the enzyme is inactivated by NEM to varying degrees depending upon the substrate or combinations of substrates used. In the presence of phenylalanine the enzyme is partially protected but both ATP and tRNA make the enzyme more susceptible to inactivation. NEM together with ATP and tRNA or all three substrates results in near-total inactivation.  相似文献   

11.
The tritium recovery assay of 9-hydroxyprostaglandin dehydrogenase [Pace-Asciak, C. (1975) J. Biol. Chem.250, 2789] has been modified to ensure its applicability to both crude and purified enzyme preparations. The stereospecificity of NAD+-dependent 9-hydroxyprostaglandin dehydrogenase with respect to NAD+ was determined first and found to be A-side specific. Based on the stereospecificity of the enzyme, a simple and sensitive assay method for 9-hydroxyprostaglandin dehydrogenase has been developed. The assay is able to detect picomole quantities of substrate conversion. When 15-keto-13,14-dihydro-[9β-3H]PGF is employed as substrate, the tritium label of the tritiated prostaglandin is effected to transfer to lactate stereospecifically by coupling 9-hydroxyprostaglandin dehydrogenase with a saturating level of lactate dehydrogenase. The amount of prostaglandin oxidized is quantitated by the radioactivity of the labeled lactate produced, which is separated from labeled prostaglandin by charcoal precipitation. Simultaneous assays with the current tritium-release and thin-layer chromatography methods indicated excellent correlation. Using this method we have found that rat kidney possesses the highest enzyme activity among those tissues examined. Rat kidney enzyme activity is linear for the first 10 min it is studied and is nonlinear with increasing amounts of crude enzyme extract, indicating the possible presence of endogenous inhibitor(s). The apparent Km for 15-keto-13,14-dihydro-PGF is 0.66 μm. The enzyme is activated by imipramine, inhibited by indomethacin, but not affected by furosemide and ethacrynic acid. These results confirm previous findings reported in the literature.  相似文献   

12.
The multifunctional enzyme complex anthranilate synthase from Neurospora crassa has been purified to homogeneity by a new procedure which yields a stable preparation of the enzyme. Unlike earlier preparations of the enzyme, anthranilate synthase prepared by this technique is not degraded during incubation at 37 °C or during freeze-thaw treatment. Purified anthranilate synthase contains two subunits of Mr 84,000 (β-subunit) and 76,000 (α-subunit), which are shown, by partial proteolysis, to be unrelated in sequence. Immunoprecipitation studies demonstrate that freshly prepared crude extracts of Neurospora contain anthranilate synthase subunits identical in size with those of the purified enzyme. The β-subunit is shown to be the product of the trp1 gene, and the a-subunit, of the trp2 gene.  相似文献   

13.
The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55–90°C, which was maximal at 80–95°C. It was highly stable within the temperature intervals 20–50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0–9.0. NaN3 and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.  相似文献   

14.
An inducible l-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe2+, and O2 for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with l-mandelate, NADPH, and ferrous sulfate and Km values for these substrates were found to be 1 × 10?4, 1.9 × 10?4, and 4.7 × 10?5m, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.  相似文献   

15.
《Insect Biochemistry》1989,19(4):435-443
Glutathione S-transferase was present in all the developmental stages of Plutella xylostella. The enzyme levels increased rapidly and reached a maximum at the pupal stage and then declined towards adulthood. The resistant strain was found to contain between 3–4 times more glutathione S-transferase than the susceptible strain. However, the enzyme from both the strains had similar Km values for GSH and DNCB, respectively. The crude enzyme had an optimum pH of 8.3 and its activity was affected by buffer molarity. The enzyme was completely inactivated on dialysis and the stability of the enzyme in the crude supernatant could be maintained in the presence of 1 mM concentrations of either GSH, 2-mercaptoethanol or cysteine. Metal ions had no effect on the stability of the enzyme. Data from Arrhenius plots, column chromatographic techniques and isoelectric focusing suggested the presence of a single form of the enzyme. The enzyme had an isoelectric point of 9.26 and a molecular weight of 36,400.  相似文献   

16.
A dipeptidase was purified to homogeneity from a crude cell extract of Streptococcus cremoris Wg2 by DEAE-Sephacel column chromatography followed by preparative disc gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 49,000. The dipeptidase is capable of hydrolyzing a range of dipeptides, but not peptides with longer chains. The enzyme was shown to be a metallo-Mn2+ enzyme with a pH optimum of 8 and a temperature optimum of 50°C. The enzyme is strongly inhibited by thiol-reducing reagents but not by sulfhydryl reagents. Kinetic studies indicated that the enzyme has a relatively low affinity for leucyl-leucine and alanyl-alanine (Km, 1.6 and 7.9 mM, respectively) but can hydrolyze these substrates at very high rates (Vmax, 3,700 and 13,000 μmol/min per mg of protein, respectively).  相似文献   

17.
Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me3)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me3)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent Km values were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me3) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me3) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis of l-carnitine from lys(Me3). The most effective inhibitors found, competitive with lys(Me3), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine. l-2-Amino-6-N-trimethylammonium-4-hexynoate, d-2-amino-6-N-trimethylammonium-4-hexynoate, and dl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd2+, Zn2+) but not by those of “hard metals” (e.g., Ca2+, Mg2+). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate, N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me3) formation. The degree of inhibition by N-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.  相似文献   

18.
Cytosolic NADP-specific isocitrate dehydrogenase was isolated from leaves of Pisum sativum. The purified enzyme was obtained by ammonium sulfate fractionation, ion exchange, affinity, and gel filtration chromatography. The purification procedure yields greater than 50% of the total enzyme activity originally present in the crude extract. The enzyme has a native molecular weight of 90 kilodaltons and is resolved into two catalytically active bands by isoelectric focusing. Purified NADP-isocitrate dehydrogenase exhibited Km values of 23 micromolar for dl-isocitrate and 10 micromolar for NADP, and displayed optimum activity at pH 8.5 with both Mg2+ and Mn2+.  相似文献   

19.
The functions of two long-chain fatty acid CoA ligase genes (facl) in crude oil-degrading Geobacillus thermodenitrificans NG80-2 were characterized. Facl1 and Facl2 encoded by GTNG_0892 and GTNG_1447 were expressed in Escherichia coli and purified as His-tagged fusion proteins. Both enzymes utilized a broad range of fatty acids ranging from acetic acid (C2) to melissic acid (C30). The most preferred substrates were capric acid (C10) for Facl1 and palmitic acid (C16) for Facl2, respectively. Both enzymes had an optimal temperature of 60 °C, an optimal pH of 7.5, and required ATP as a cofactor. Thermostability of the enzymes and effects of metal ions, EDTA, SDS and Triton X-100 on the enzyme activity were also investigated. When NG80-2 was cultured with crude oil rather than sucrose as the sole carbon source, upregulation of facl1 and facl2 mRNA was observed by real time RT-PCR. This is the first time that the activity of fatty acid CoA ligases toward long-chain fatty acids up to at least C30 has been demonstrated in bacteria.  相似文献   

20.
B. Halliwell 《FEBS letters》1983,151(2):313-316
Glycerate kinase from spinach leaves was purified to near homogeneity using PEG/MgCl2 fractionation, ion exchange, molecular sieving and affinity chromatography. The purified enzyme is a monomer of Mr 40 000, shows a pI-value of 4.8 and a broad pH optimum of 6.5–8.5 and is specific for D-isomer of glycerate. The high activity of crude enzyme (≈ 150 μmol. h?1.mg chl?1) indicates that glycerate kinase does not limit the oxidative photosynthetic carbon cycle.  相似文献   

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