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1.
Mostafa H. Mostafa Donald J. Hanahan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(3)
Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at −20°C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide. 相似文献
2.
Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth 总被引:4,自引:3,他引:1
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Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the Kmapp of arginine decarboxylase for its substrate and reduced the Vmaxapp by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed. 相似文献
3.
Mostafa H. Mostafa Donald J. Hanahan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(3):490-500
Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at ?20°C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide. 相似文献
4.
A strain of Thermomyces lanuginosus, isolated from hot spring water in Turkey, was studied for optimization of phytase production using solid-state fermentation. Effects on fermentation of different production parameters such as substrate type, moisture, culture time, and inoculum size were investigated using a one-factor-at-a-time approach. Central composite design (CCD) of response surface methodology was applied for the optimization of four factors (culture temperature, initial pH, aeration area, age of seeding culture) that were affecting phytase production by Thermomyces lanuginosus in rice bran. Maximum phytase activity was achieved by using rice bran. The optimum levels of variables that supported maximum enzyme activity were moisture 70%, culture time 7 days, inoculum size 40%, culture temperature 55°C, initial pH 7.5, aeration area 30%, age of seeding culture 5 days, sucrose 1%, and ZnSO4 2.5 mM. An overall 10.83-fold enhancement in phytase activity (0.30 to 3.248 U) was attained due to the optimization. 相似文献
5.
Summary A 0.1% Triton X-100 extract of human erythrocyte plasma membranes contained high proteolytic activity as determined by a very sensitive assay utilizing3H-acetylated hemoglobin (162 cpm/pmole) as a substrate. Two proteolytic enzymes having optimum activity at pH 3.4 and pH 7.4 were isolated from Sephadex G-100. The protease active at pH 3.4 was 75 times as active as the pH 7.4 enzyme and it was purified 182-fold over the original homogenate and characterized. A linear relationship for activity versus time and activity versus concentration of enzyme was found. The optimum temperature was 37°C and theK
m
was 1×10–5
m hemoglobin. No enzyme activation was observed with any cation studied and EDTA had no inhibitory effect; (10mm Fe+3 and Hg+2 were inhibitory). The pH 3.4 protease was stable indefinitely at –20°C in 0.1% Triton X-100. Gel electrophoresis was performed on a sodium dodecylsulfate-mercaptoethanol enzyme preparation and two protein bands (mol. wt. 33,000 and 54,000) were evident for the Sephadex G-200 eluate containing the pH 3.4 protease. 相似文献
6.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(5):911-915
In this study, glutathione S-transferase (GST) enzyme was purified from nontumour and tumour human gastric tissue and in vitro effects of heavy metals on the enzyme were examined. GST was purified 3089 fold with a specific activity of 20 U/mg and a yield of 78% from gastric tumour tissue; and 1185 fold with a specific activity of 5.69 U/mg and a yield of 50% from nontumour tissue by using glutathione?agarose affinity column, respectively. Enzyme purity was verified by SDS-PAGE and subunit molecular mass was calculated around 26 kDa. The molecular weight of the enzyme was calculated as 52 kDa by using Sephadex G-75 gel filtration column. Then, inhibitory effects of metal ions on the enzymes were investigated. Mg2+ and Cd2+ had inhibitory effect on the enzymes activities. Other kinetic properties of the enzymes were also determined. 相似文献
7.
Enhancement and stabilization of the production of glucoamylase by immobilized cells of Aureobasidium pullulans in a fluidized-bed reactor 总被引:1,自引:0,他引:1
Federico Federici Maurizio Petruccioli Martin W. Miller 《Applied microbiology and biotechnology》1990,33(4):407-409
Summary Glucoamylase production by Aureobasidium pollulans A-124 was compared in free-living cells, cells immobilized in calcium alginate gel beads aerated on a rotary shaker (agitation rate 150 rpm), and immobilized cells aerated in an air bubble column reactor. Fermentation conditions in the bioreactor were established for bead concentration, substrate (starch) concentration, calcium chloride addition to the fermentation medium, and rate of aeration. Production of glucoamylase was optimized at approximately 1.5 units of enzyme activity/ml medium in the bioreactor under the following conditions: aeration rate, 2.0 vol air per working volume of the bioreactor (280 ml) per minute; gel bead concentration, 30% of the working volume; substrate (starch) concentration, at 0.3% (w/v); addition of calcium chloride to the medium at a final concentration of 0.01 M. Productivity levels were stabilized through the equivalent of ten batches of medium with the original inoculum of immobilized beads.
Offprint requests to: M. Petruccioli 相似文献
8.
Irmgard Ziegler 《Phytochemistry》1974,13(11):2411-2416
SO32? acts on NAD- and NADP-dependent malate dehydrogenase in several ways. Firstly, SO32? favours the appearance of low MW species (65 000 and 39 000 daltons) in Sephadex gel chromatography. Secondly, the enzyme form which is obtained by gel chromatography with dithioerythritol plus nucleotide cofactor is changed in the presence of SO32?. This is indicated by the appearance of a linear reaction (instead of curvilinear), and by the abolition of the biphasic sigmoidal kinetics on varying substrate and cofactor concentrations. SO32? causes the loss of negative cooperativity at low substrate or cofactor concentrations. Thus the inhibition of initial velocity at high substrate or cofactor concentrations is even more marked than at lower ones. Thirdly, SO32? strongly reduces the activity in substrate saturating conditions. 相似文献
9.
Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase 总被引:4,自引:0,他引:4
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Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The Km value was found to be 0.48 × 10−4 M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels. 相似文献
10.
Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo
José Peinado Javier Florindo J. López-Barea 《Molecular and cellular biochemistry》1992,110(2):135-143
Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH
or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in
starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to
redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation
of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate
or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation
was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP+ concentrations.
The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144%
and NADP+ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity
descended to 23% of the control value, while the NADH/NAD+ and NADPH/NADP+ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential
of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase. 相似文献
11.
The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease
from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation
chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide
gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein
as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation.
It was strongly activated by metal ions such as Ca2+, Mg+2, and Mn+2. Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP)
but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the
presence of 10mM CaCl2 and 1M glycine. The addition of 10mM CaCl2 and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it
retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood
stains completely when used with detergents in the presence of 10mM CaCl2 and 1M glycine. 相似文献
12.
Summary Partially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10–4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10–5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect. 相似文献
13.
Production of tannase (tannin acyl hydrolase, EC 3.1.1.20) by Aspergillus nigerHA37 on a synthetic culture medium containing tannic acid at different concentrations has been studied. Maximal enzymatic activity increased according to the initial concentration of tannic acid; respectively 0.6, 0.9 and 1.5 enzyme activity units (EU) ml−1 medium in the presence of 0.2%, 0.5% and 1% of tannic acid. Tannase production by A. niger HA37 on fourfold diluted olive mill waste waters (OMWW) as substrate, was between 0.37 and 0.65 EU ml−1. Enzyme production on the diluted OMWW remained globally stable during more than 30 h. Growth of A. niger HA37 on OMWW was correlated with about 70% degradation of phenolic compounds present in the waste. This strain has therefore the capacity to degrade complex wastewaters which cause environmental damage to aquatic streams. 相似文献
14.
Total and specific activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT) varied widely among six tissues from C3H/f mice; the highest levels of activity were in brain. More striking were thermostability differences in tissue enzymes. Although brain, spleen, and kidney HPRT retained 65% basal activity after 15 min at 85 C, heart, liver, and erythrocyte HPRT retained only 20–30% initial activity. Kidney HPRT behaved as monospecific heat-stable enzyme (K
denaturation=0.022/min, and liver enzyme behaved as monospecific heat-labile enzyme (K
denaturation=0.061/min), while other tissues appeared to contain both forms of the enzyme. Multiple electrophoretic activity bands were present in all tissues; no activity band was restricted to a single tissue. The data presented here are consistent with the hypothesis that the distinct tissue properties of HPRT result from posttranslational modification of the product of a single genetic locus which is expressed in all tissues.This study was supported in part by NIH Grant AM 16722 and by an Institutional Biomedical Grant. 相似文献
15.
An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-14C as substrate and radioassaying the 14CO2 evolved. Mn2+ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%. 相似文献
16.
Effects of aeration and of corn steep concentration on L-asparaginase production in Escherichia coli
J. Netrval 《Applied microbiology and biotechnology》1980,9(3):185-187
Summary The production of L-asparaginase was investigated in Escherichia coli, growing under different conditions of aeration in a medium containing 2% or 6% corn steep. At both concentrations, excessive aeration decreased enzyme production. In the medium with 2% corn steep, L-asparaginase activity began to decline as soon as the oxygen absorption exceeded 0.22 mmol O2 l–1 min–1, and when the oxygen absorption rate was 1.26 mmol O2 l–1 min–1, enzyme activity reached only about 5% of maximum. In the medium with 6% corn steep, a decline of L-asperaginase activity did not appear until the oxygen absorption rate value exceeded 0.54 mmol O2 l–1 min–1, at the oxygen absorption rate of 1.26 mmol O2 l–1 min–1, the enzyme activity still reached about 50% of maximum. 相似文献
17.
Shuwsei Kamimiya Tsuguaki Nishiya Kazuo Izaki Hajime Takahashi 《Bioscience, biotechnology, and biochemistry》2013,77(5):1071-1078
A strain of Erwinia aroideae produces a remarkable amount of pectolytic enzyme when the organism was induced by nalidixic acid for the bacteriocin production. This pectolytic enzyme was purified approximately 60-fold from the induced medium by carboxymethyl-cellulose and Sephadex G–75 gel column chromatographies after batchwise treatment with carboxymethyl- and diethylaminoethyl-celluloses. The purified enzyme was almost homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a molecular weight of about 28,000 to 32,000 was determined for this enzyme. The optimum pH of the enzyme activity was about 8.0 to 8.2. The purified enzyme produced reaction products from pectin and methoxylated pectic acid which had a strong absorption at 235 nm indicating a trans-eliminase reaction. Pectin or pectic acid with higher methoxyl content was a good substrate for this enzyme, while no significant activity was observed when pectic acid was a substrate. The limit of degradation of pectin and pectic acid with higher methoxyl content (90% esterified) by the enzyme were 6.5% and 43%, respectively. It was concluded that the enzyme is a new endo-pectin trans-eliminase from bacterial origin. 相似文献
18.
M G Fortina A Acquati P Rossi P L Manachini C Di Gennaro 《Journal of industrial microbiology & biotechnology》1996,17(2):69-72
After induction, seven strains ofBotrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L–1 of glucose and 2 g L–1 of yeast extract at pH 3.5, 2 g L–1 gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24°C. By optimizing the culture conditions, the enzyme activity reached 28 U ml–1 in 5 days with a specific activity of 560 U mg–1 protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40°C, respectively, with syringaldazine as the substrate. 相似文献
19.
Abstract— Bovine pineal gland S-adenosylmethionine: N-acetylserotonin O-methyltransferase has been purified about 2800-fold using cell fractionation, ammonium sulphate treatment, Sephadex G-200 gel filtration and anion exchange chromatography. The enzyme has been found to be a polymer; the smallest unit observed had a mol. wt. of 21,800 and the other polymers' molecular weights were multiples of this figure. In the gland extract polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. were more abundant than the others; they showed also higher specific activity. One of the products of the reaction, S-adenosylhomocysteine was found to be a potent inhibitor, whereas the other product, melatonin, did not inhibit the bovine pineal gland enzyme, even at much higher concentrations. Homocysteic acid, cysteic acid, GSG and GSSG inhibited the enzyme. The required concentrations for this effect was 100 times higher than that of S-adenosylhomocysteine. The addition of GSH to the medium during purification led to complete loss of activity. Adenosine, homocysteine and other thio compounds had little or no effect. The enzyme was found to be activated by its substrates and also by certain anions. Among various organic acid salts, citric acid cycle intermediates were found to be good activators; their nonsubstituted analogues were not as effective. The activator effect of oxaloacetate and bicarbonate was the highest, and was brought about by relatively low concentrations of these anions (1–5 × 10?3 M), hence their effect was considered specific. The degree of activation caused by oxaloacetate was decreased by increasing substrate concentrations and vice versa. The S-adenosylhomocysteine inhibition could not be reduced by increasing the substrate concentration; S-adenosylhomocysteine also inhibited the oxaloacetate-activated enzyme. These observations have been explained by the allosteric behaviour of the enzyme. The kinetic behaviour of various polymers was also investigated. The highest substrate and oxaloacetate activation and the highest S-adenosylhomocysteine inhibition was observed for polymers of 83,000, 100,000, 125,000 and 150,000 mol. wt. The Km values for S-adenosylmethionine and N-acetylserotonin calculated for the oxaloacetate activated enzyme were also lower for these polymers than others. 相似文献
20.
Irmgard Ziegler 《Phytochemistry》1974,13(11):2403-2410
Sephadex G-200 gel filtration of an ammonium sulfate fraction, containing the bulk of NAD-dependent malate dehydrogenase, yields forms of differing MW. Both Mg2+ and NADH stabilize the 127000 daltons MW form. K+, or incubation with dithioerythritol, cause splitting and partial reaggregation, resulting in MWs ranging between 35000 and 180000 daltons. Chromatography in the presence of dithioerythritol and NADH results in an enzyme with a non-linear reaction rate at low substrate concentrations. Plots of initial velocity vs substrate and cofactor concentration respectively are characterized by two slopes of positive cooperativity separated by an intermediary plateau of negative cooperativity. Gel chromatography in the presence of Mg2+ or K+ or drastic dilution of the enzyme results in an enzyme with linear reaction rates also at low substrate concentration. Its kinetics are consistent with the view that the enzyme undergoes conformational changes when the substrate concentration is varied. 相似文献