共查询到20条相似文献,搜索用时 15 毫秒
1.
《Journal of Fermentation and Bioengineering》1991,71(2):134-136
Methylglyoxal reductase was purified from Hansenula mrakii IFO 0895 to a homogenous state on polyacrylamide gel electrophoresis. The enzyme consisted of a single polypeptide chain with a molecular weight of 34,000. The enzyme was specific to methylglyoxal (Km = 1.92 mM) and NADPH (Km = 40.8 μM). The activity of the enzyme was inhibited by p-chloromercuribenzoate and HgCl2. NADP also inhibited the activity of the enzyme, and the Ki value was calculated to be 0.25 mM. 相似文献
2.
《Plant science》1987,51(1):9-16
A secreted argnase (molecular weight 245 000) from Evernia prunastri thallus has been purified 1480-fold from media in which the thalli were incubated for 8 h in the dark. The enzyme is a glycoprotein which contains 280 residues of glucose, 27 of fructose and 85 of mannose per molecule. The Km-value of the enzyme has been estimated as 1.5 mM for L-arginine, with an interaction coefficient of nH ≅ 1, calculated from Hill plot. The enzyme is activated by D-usnic acid, the only phenol which appears in the incubation media. This phenol behaves as a non-essential activator of the enzyme with a Ka-value of 0.19 mM. 相似文献
3.
The rate of O2 uptake and the activity of NAD-specific isocitrate dehydrogenase (NAD-ICDH) of mitochondria isolated from castor bean cotyledons were increased by added cis, trans-abscisic acid (ABA) in an in vitro system, while the NADP-specific isocitrate dehydrogenase (NADP-ICDH) was not affected by cis, trans-ABA. Trans, trans-ABA showed only a slightly inhibitory effect on O2 uptake. The Vmax value for the isotherm of isocitrate by the enzyme was also increased by cis, trans-ABA. The isocitrate Km value for the enzyme with cis, trans-ABA was calculated to be approximately 249.8 micromolar, while the S0.5 for the enzyme without the ABA was 151.6 micromolar. The n value calculated from the slopes of Hill plots of the reaction velocity of NAD-ICDH against isocitrate concentration was 1.5 in the mitochondrial fraction in the absence of ABA, and cis, trans-ABA treatment decreased the value to 1.0. Cis, trans-ABA also partly overcame the inhibition of NAD-ICDH activity by ATP. 相似文献
4.
S Asami K Inoue K Matsumoto A Murachi T Akazawa 《Archives of biochemistry and biophysics》1979,194(2):503-510
NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C4 pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s20,w) of 9.7 and molecular weight of 2.27 × 105 in sucrose density gradient centrifugation, and molecular weight of 2.26 × 105 calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 104 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO3? proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: Km (malate), 0.4 mm; Km (NADP), 17.6 μm; Km (Mg2+), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol. 相似文献
5.
《Journal of Fermentation and Bioengineering》1991,71(2):131-133
Glyoxalase I was purified from Hansenula mrakii IFO 0895 which was resistant to 25 mM methylglyoxal. The molecular weight of the purified enzyme was calculated to be 38,000 by both gel-filtration of Sephadex G-150 and SDS-PAGE. The enzyme was almost specific to methylglyoxal (Km = 0.91 mM). The activity of the enzyme was not inhibited by metal ion chelators such as EDTA, which is a potent inhibitor for glyoxalase Is from other sources. 相似文献
6.
Estimating Kinetic Parameters when the Amount of Enzyme Added to an Assay Is Not a Controlled Variable: NITROGENASE ACTIVITY OF INTACT LEGUMES
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Davis LC 《Plant physiology》1980,66(1):126-129
Reliable estimates of Michaelis constants (Km) and inhibitor constants may be obtained, in the absence of control over the amount of enzyme being added to any assay system, provided the following constraints are met. Michaelis-Menten kinetics are obeyed. Two rate measurements must be made with the same sample of enzyme: at low and high substrate concentration for determining Km or minus and plus an inhibitor for determining inhibitor constants. The Michaelis constant may be calculated from the equation [Formula: see text] Inhibitor constants are derived graphically from Lineweaver-Burk or Dixon plots, once the Km has been calculated. The above technique has been applied to study of the acetylene-reducing ability of intact legume plants. The apparent Km for acetylene reduction by nitrogenase in legume nodules is ~1/100 atmosphere in the absence of nitrogen and ~1/40 atmosphere in its presence. 相似文献
7.
A series of N-alkylmaleimides, varying in chain length from N-ethylmaleimide and N-butyl to N-octyl, inclusive, was shown to effectively inactivate rat ovarian 20α-hydroxysteroid dehydrogenase at pH 7.7, 25 °C. The apparent second-order rate constants for inactivation were observed to increase with increasing chain length of the N-alkylmaleimide used. Positive chain length effects were also indicated by the Kd values for N-alkylmaleimides calculated from double-reciprocal plots resulting from the saturation kinetics observed in the inactivation reactions. The maximum rate constant for inactivation at enzyme saturation was 0.3 min?1 for each maleimide studied. NADP-and coenzyme-competitive inhibitors such as 3-aminopyridine adenine dinucleotide phosphate and various adenosine derivatives protected the enzyme against maleimide inactivation, whereas no protection was observed with the steroid substrate, 20α-hydroxypregn-4-en-3-one. The pH profile for maleimide inactivation indicated the involvement of an enzyme functional group with a pKa near 8.0. Sulfhydryl modification was also indicated by fluorescein mercuric acetate inactivation and titration experiments. Inactivation of the enzyme by a lysine-modifying reagent exhibited a pH profile differing from that observed in the maleimide inactivation process. It is proposed that N-alkylmaleimides inactivate the enzyme through covalent modification of sulfhydryl groups located in a nonpolar region of the enzyme. 相似文献
8.
Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues 总被引:3,自引:3,他引:0
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The Km values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP+ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP+ oxidoreductase. The effect of NaCl and MgCl2 concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP+ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues. 相似文献
9.
《Experimental mycology》1989,13(3):294-298
Succinate dehydrogenase (EC 1.3.99.1) fromDictyostelium discoideum was purified 40-fold. The pH optimum for the reaction underin vitro conditions was 7.4. Divalent cations showed no effect on the enzyme activity. Lineweaver-Burk plots of initial velocity data were linear. The Km value for succinate was calculated to be 0.22 mM. Apparent Ki values for fumarate, malonate, and oxaloacetate were 0.4, 0.02, and 0.003 mM, respectively. All three showed a competitive inhibition pattern. A comparison of the reaction ratein vivo with the calculated enzyme activity requiredin vivo (Vv) suggests that succinate dehydrogenase may be rate controlling to flux through the citric acid cycle. 相似文献
10.
《Process Biochemistry》1999,34(4):399-405
Cyclodextrin glucosyltransferase from Paenibacillus macerans NRRL B-3186 was immobilized on aminated polyvinylchloride (PVC) by covalent binding with a bifunctional agent (glutaraldehyde). The immobilized activity was affected by the length of the hydrocarbon chain attached to the PVC matrix, the amount of the protein loaded on the PVC carrier, and glutaraldehyde concentration. The activity of the immobilized enzyme was 121 units/gram carrier, the specific activity calculated on bound protein basis was 48% of the soluble enzyme. Compared to the free enzyme, the immobilized form exhibited: a higher optimal reaction temperature and energy of activation, a higher Km (Michaelis constant) and lower Vmax (maximal reaction rate), improved thermal stability and resistance to chemical denaturation. The operational stability was evaluated in repeated batch process and the immobilized enzyme retained about 85% of the initial catalytic activity after being used for 14 cycles. 相似文献
11.
Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn2+ for activity. Maximal activity was at 1.5 millimolar MnCl2. A Km of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP-dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride. 相似文献
12.
Malonyl-CoA decarboxylase (EC 4.1.1.9) was found to be localized in the mitochondria in rat liver. Low ionic strength (10 mm Na phosphate) buffer extracted the bulk (>85%) of the enzyme from the mitochondria. From this extract the enzyme was purified over 2,000-fold using a combination of (NH4)2SO4 precipitation, gel filtration with Sepharose 4B and Sephadex G-150, ion exchange chromatography with QAE-Sephadex and CM-Sephadex, and finally chromatography on NADP-agarose. The purified enzyme, which had a specific activity of about 16 μmol/min/mg, appeared to be electrophoretically homogeneous and had a molecular weight of 160,000. The decarboxylase had a broad pH optimum between 8.5 and 10.0 and showed a typical Michaelis-Menten substrate saturation pattern from which Km and V were calculated to be 54 μm and 18.8 μmol/min/mg, respectively. This enzyme decarboxylated neither malonic acid nor methylmalonyl-CoA and was severely inhibited by thiol-directed reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide but not by iodoacetamide. Acetyl-CoA, propionyl-CoA, and methylmalonyl-CoA also inhibited the enzyme. The purified decarboxylase was immunogenic in rabbits and Ouchterlony double diffusion analysis revealed a single precipitant line with the purified enzyme. The IgG fraction isolated from the antiserum inhibited the enzyme from not only liver mitochondria but also the mammary gland, heart, and kidney of the rat. However, malonyl-CoA decarboxylase from rat brain mitochondria was not inhibited by the antibody. Malonyl-CoA decarboxylase purified from the uropygial gland of a domestic goose neither cross reacted nor was it inhibited by the antiserum prepared against the rat liver mitochondrial enzyme and the antibody against the goose enzyme neither cross-reacted nor inhibited the enzyme from the rat. It is proposed that a role for mitochondrial malonyl-CoA decarboxylase is to decarboxylate malonyl-CoA generated by propionyl-CoA carboxylase and thus protect mitochondrial enzymes susceptible to inhibition by malonyl-CoA. 相似文献
13.
Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases 总被引:7,自引:5,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purified from two cyanobacteria: the filamentous, heterocystic, Anabaena PCC 7120 and the unicellular Synechocystis PCC 6803. The purification procedure gave highly purified enzymes from both cynobacteria with specific activities of 134 (Synechocystis) and 111 (Anabaena) units per milligram protein. The purified enzymes migrated as a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular mass corresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons. Tetrameric structures were determined for the native enzymes by analysis of gel filtrations. Kinetic and regulatory properties were characterized for the cyanobacterial ADPglucose pyrophosphorylases. Inorganic phosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. The Synechocystis enzyme was activated 126-fold by 3-phosphoglycerate, with saturation curves exhibiting sigmoidicity (A0.5 = 0.81 millimolar; nH = 2.0). Activation by 3-phosphoglycerate of the enzyme from Anabaena demonstrated hyperbolic kinetics (A0.5 = 0.12 millimolar; nH = 1.0), having a maximal stimulation of 17-fold. I0.5 values of 95 and 44 micromolar were calculated for the inhibition by inorganic phosphate of the Synechocystis and Anabaena enzyme, respectively. Pyridoxal-phosphate behaved as an activator of the cyanobacterial enzyme. It activated the enzyme from Synechocystis nearly 10-fold with high apparent affinity (A0.5 = 10 micromolar; nH = 1.8). Phenylglyoxal modified the cyanobacterial enzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showed that anti-spinach leaf (but not anti-Escherichia coli) ADPglucose pyrophosphorylase serum inactivated the enzyme from cyanobacteria. When the cyanobacterial enzymes were resolved on sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoresis and probed with Western blots, only one protein band was recognized by the anti-spinach leaf serum. The same polypeptide strongly reacted with antiserum prepared against the smaller spinach leaf 51 kilodalton subunit, whereas the anti-54 kilodalton antibody raised against the spinach subunit reacted weakly to the cyanobacterial subunit. Regulatory and immunological properties of the cyanobacterial enzyme are more related to the higher plant than the bacterial enzyme. Despite this, results suggest that the ADPglucose pyrophosphorylase from cyanobacteria is homotetrameric in structure, in contrast to the reported heterotetrameric structures of the higher plant ADPglucose pyrophosphorylase. 相似文献
14.
Studies on a gram-positive hydrogen bacterium,Nocardia opaca 1 b 总被引:7,自引:0,他引:7
Nocardia opaca strain 1 b has a NAD-dependent hydrogenase (hydrogen dehydrogenase). The enzyme has been purified from autotrophically grown cells and tested for optimal assay conditions and stability. The purification procedure involved protamine sulfate treatment, ammonium sulfate precipitation, and separation by DEAE-cellulose and Sephadex G-200 chromatography and resulted in a 63-fold increase of specific activity at a 11.7% enzyme recovery. The final specific activity was 103 μmoles H2/min·mg protein. The purified enzyme was dependent on nickel and magnesium ions at 0.5 and 5.0 mM concentrations, respectively, as well as flavin mononucleotide at a 5–10 μM concentration. Straight enzyme kinetics were achieved by preincubating the enzyme in the presence of NADH2. A high stability of the enzyme was observed in 0.1 M potassium phosphate buffer, pH 6.5, in the presence of 0.5 mM nickel and 5 mM magnesium ions under hydrogen atmosphere. Even under air the enzyme was remarkably stable, although less than under hydrogen. From double reciprocal plots of substrate saturation curves the Michaelis-Menten constants were calculated: For saturating NAD-concentration the K m was 0.063 mM H2 and for saturating hydrogen concentration the K m was 0.123 mM NAD. 相似文献
15.
Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The Km value toward glycylprolyl-p-nitroanilide is about 6.0 × 10?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg2+, Pb2+, Zn2+, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors. 相似文献
16.
《The International journal of biochemistry》1981,13(7):865-869
- 1.1. Sedimentation velocity and sedimentation equilibrium studies of bovine heart AMP-deaminase were performed. Molecular weights of the native enzyme and subunit were determined as 161,000 and 43,000 dallons respectively.
- 2.2. The kinetic data indicate that in the presence of 100 mM KCl the enzyme may be active as a dimer.
- 3.3. The influence of temperature on the enzyme kinetics was investigated, from which activation energy (Ea and the heat of enzyme-substrate complex formation (ΔHs) were calculated.
- 4.4. It is suggested that an equilibrium may exist between a dimeric and tetrameric form of AMP-deaminase in the heart.
17.
Yakup Kolcuoğlu 《Process Biochemistry》2012,47(12):2449-2454
Polyphenol oxidases (PPO) are very important enzymes group in many industrial applications, especially in food, medicine and cosmetics. PPO from Macrolepiota gracilenta, a wild edible mushroom, was purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column and characterized in terms of mono- and diphenolase activity. The highest activities for pure enzyme were observed in the presence of PHPPA and DHPPA for monophenolase and diphenolase, respectively. The enzyme showed pH optimum values at 7.0 and 5.0, respectively, for monophenolase and diphenolase activities. Km values calculated as 0.8 mM for monophenolase and 1 mM for diphenolase activity at the presence of PHPPA and DHPPA as substrate, respectively. Vmax values were calculated as 2000 U/mg protein for both activity. Monophenolase and diphenolase activities were conserved approximately 40% and 60%, respectively, in their optimum pH at 4 °C after 5 day incubation. The activities were inhibited most effectively by thiourea. The data obtained from this study showed that this enzyme could be useful for some industrial purposes. 相似文献
18.
19.
A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-100 and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K m), maximal reaction velocity (V max) and turnover number (K cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s?1, respectively. The best enzyme activators were K+, Ca2+ and Mn2, respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry. 相似文献
20.
J Hoebeke J Groswasser N Bousard E Vamos A D Strosberg 《Archives of biochemistry and biophysics》1979,198(2):556-561
The temperature dependence for the hydrolysis of both 4-methylumbelliferyl-α-l-fucoside and p-nitrophenyl-α-l-fucoside was determined for purified α-l-fucosidase (EC 3.2.1.51) from human placenta. The inhibition of the enzymatic reaction by l-fucose was also studied using the first of these two substrates at different temperatures. The thermodynamic parameters calculated from the pKm were for the 4-methylumbelliferyl-conjugate ΔF = ?6.6 kcal/mol, ΔH = ?8.5 kcal/mol, and ΔS = ?6.3 e.u. and for the p-nitrophenylconjugate ΔF = ?5.6 kcal/mol, ΔH = ?12.2 kcal/mol, and ΔS = ?21.1 e.u. The thermodynamic parameters for l-fucose were ΔH = ?12.4 kcal/mol and ΔS = ?20.1 e.u. The lower exothermicity and negative entropy calculated for the 4-methylumbelliferyl substrate compared to the thermodynamic parameters calculated for the p-nitrophenyl substrate and l-fucose suggest the existence of a secondary hydrophobic binding site for the 4-methylumbelliferyl moiety on the enzyme. The difference in the enthalpy for both substrates is also reflected in a difference in activation energy, being 15.8 kcal/mol for the 4-methylumbelliferyl substrate and 20.7 kcal/mol for the p-nitrophenyl substrate. From these results it may be concluded that altered kinetic properties of the enzyme could be the result of the binding of the “aglycone” moiety of the fluorogenic substrate to the enzyme. 相似文献