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1.
Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations. 相似文献
2.
Barbaros Dinçer Arife Pınar Ekinci Gülay Akyüz İlker Zeki Kurtoğlu 《Journal of enzyme inhibition and medicinal chemistry》2016,31(6):1662-1665
An α-carbonic anhydrase (CA, EC 4.2.1.1) was purified and characterized kinetically from gill of Acipenser gueldenstaedtii as an endangered sturgeon species. The carbonic anhydrase was purified 66-folds with yield 20.7% by Sepharose-4B-l-tyrosine-sulfanilamide affinity column and the specific activity was determined as 222.2?EU/mg protein. Km and Vmax kinetic values for gill carbonic anhydrase were calculated by a Lineweaver–Burk graph using p-nitrophenol acetate (p-NPA) as a substrate, and was defined as 2.5?mM and 5?×?106?μM/min, respectively. It was observed that CA from the sturgeon gill in the presence of the sulfanilamide and acetazolamide as an inhibitor had very low IC50 values such as 13.0 and 0.1?μM, respectively. In addition, it was determined that the enzyme was inhibited by Fe2+, Co2+, Ni2+, and Zn2+–Ba2+ with the IC50 values of 0.2, 1.7, 1.2, and 1.1?mM, respectively. 相似文献
3.
Yasemin Camadan Hasan Özdemir 《Journal of enzyme inhibition and medicinal chemistry》2016,31(6):1335-1341
Dihydropyrimidine dehydrogenase (DPD, E.C. 1.3.1.2) was purified from sheep liver with a yield of 16.7%, purification fold of 407.5 and specific activity of 0.705?EU/mg proteins. The purification procedure consisted of ammonium sulphate fractionation, DEAE ion exchange chromatography and 2′,5′-ADP Sepharose-4B affinity chromatography. The molecular weight determined by SDS-PAGE and was found 111?kDa. Optimum pH, ionic strength temperature and stable pH were determined as 8.0, 0.9?mM, 50?°C and 6.0, respectively. The kinetic parameters (Km and Vmax) of the enzyme were determined with NADPH as 22.97?μM and 0.17?EU/mL, respectively. The same parameters were determined with uracil as 17.46?μM and 0.14?EU/mL, respectively. Additionally, in vitro inhibitory effects of some antidepressant drugs including escitalopram, fluoxetine, mirtazapine, haloperidol and some anaesthetic drugs including propofol and lidocaine were investigated against DPD. In addition, IC50 values for each active drug obtained for escitalopram, fluoxetine, mirtazapine, haloperidol, propofol and lidocaine were determined as 1736.11, 13.24, 86.65, 99.03, 0.21 and 15.07?μM, respectively. 相似文献
4.
Rishiram Ramanan Krishnamurthi Kannan Nadimuthu Vinayagamoorthy Kunga Mohan Ramkumar Saravana Devi Sivanesan Tapan Chakrabarti 《Biotechnology and Bioprocess Engineering》2009,14(1):32-37
Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its
mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide
affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass
of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme
revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn2+, Co2+, Cu2+, Fe3+, Mg2+, and anion SO4− increased enzyme activity while strong inhibition was observed in the presence of Hg2+, Cl−, HCO3−, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme
kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the Vmax and Km values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The Ki value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing
imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms. 相似文献
5.
Hamed Doostdar T.Gregory McCollum Richard T Mayer 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,118(4):861-867
An endo-polygalacturonase (PG) (EC:3.2.1.15) with a pI of 9.4 and an Mr of 44,500 was purified to electrophoretic homogeneity from the gut of West Indies sugarcane rootstalk borer weevil (Diaprepes abbreviatus L.) larvae. Hydrolytic activity was maximal in 150 mM sodium acetate, pH 5.5, at 30°C. Kinetic determinations yielded an apparent Km of 3.68 mg polygalacturonic acid (PGA)/ml and a Vmax of 283 μmol galacturonic acid/min/mg protein for PGA. Enzymatic activity was inhibited by a polygalacturonase inhibitor protein from “Hamlin” orange flavedo. The purified protein does not appear to be glycosylated, and its N-terminal sequence showed no homology to any PG protein sequences in data banks. 相似文献
6.
R. Ramapriya A. Thirumurugan T. Sathishkumar D.R. Manimaran 《Journal of Genetic Engineering and Biotechnology》2018,16(2):363-367
Inulinase are industrial food enzymes which have gained much attention in recent scenario. In this study, Inulinase producing eight bacterial colonies were isolated and screened from three different plant root tubers soil sample. Among 8 inulinase producing colonies, the higher yielding colony was selected with 25.10?U/mL for further studies. The best inulinase producing colony was identified by partial 16S rRNA gene sequence as Bacillus sp. The crude inulinase was purified by using ammonium sulphate precipitation, dialysis and ion exchange chromatography on DEAE – sephacel and obtained 1.9 purification fold with total activity 293 U. The purified enzyme was subjected to characterization studies and it was found to be stable at 30–60?°C and optimum temperature was at 55?°C. The enzyme was stable at pH 3.0–7.0 and optimum pH was at 6.5. The Km and Vmax value for inulinase was found to be 0.117?mg/mL and 4.45?μmol?min?mg?1 respectively, demonstrate its greater affinity. Hence, this enzyme can be widely used for the production of fructose, and fructooligosaccharides, which are important ingredients in food and pharmaceutical industry. 相似文献
7.
A simple procedure for the isolation of cathepsin-B from bovine pancreas employing ammonium sulphate fractionation, DEAE cellulose
chromatography and Sephadex G-200 gel filtration is described. The purified enzyme gave a single band on polyacrylamide gel
electrophoresis. The molecular weight as determined by gel filtration of the enzyme was 26,850. ItsK
m andV
max values were 12.8 mM and 0.303 Μmol/min/mg, respectively. TheK
i for iodoacetamide was 0.16 mM. 相似文献
8.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1247-1251
Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50,000 by both Sephadex G-100 gel filtration and SDS–polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitrophenylphosphorylcholine. Vmax and Km values were 5.0, μmol/min/mg protein and 0.3 mM, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca2+ -dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250, μM Ca2+ and higher Ca 2+ concentrations than 1 mM had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca2+ concentrations in the rice cells is discussed. 相似文献
9.
Four isoenzymes of carbonic anhydrase (CA) were purified from Elephas Irogontherii (steppe elephant) bone (approx 0.3-0.5 million years old) from different locations (outer peripheral, cytosolic, inner peripheral and integral) using Sepharose 4B-L-tyrosine sulphanilamide affinity chromatography and their kinetics properties were investigated and compared with known CA isoenzymes. The purification degree of CAs was monitored by SDS-PAGE. Purification fold for outer peripheral, inner peripheral, cytosolic and integral CA was 395.6, 652.8, 1091 and 429.3 and the molecular mass (as determined by gel filtration chromatography) was 37, 36, 35, and 39 kDa, respectively. The optimal temperature for isozymes was 10-20, 30, 30 and 60 degrees C and optimal pH- was between 7.5-11, 7.5-10, 7.5-10 and 7.5 respectively. K(m) values (at optimum pH and 20 degrees C) for p-nitrophenyl acetate as substrate were 4.83, 6.80, 4.525 and 3.86 mM and the Vmax values for the same substrate were 0.00097, 0.0149, 0.00249 and 0.00072 micromol/L*min, respectively. I50 values of isoenzymes for the inhibitors of CA - sulphanilamide, KSCN, acetazolamide and NaN3 were also determined. 相似文献
10.
M. Vanko D. Rauová L. Bezáková I. Holková F. Bilka M. Cupáková 《Biologia Plantarum》2012,56(1):105-110
Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography
on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined
by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic
acid (KM = 1.78 mM and Vmax = 11.4 μmol mg−1 min−1) and linolenic acid (KM = 1.27 mM and Vmax = 10.2 μmol mg−1 min−1). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products
revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products
being about 1:1. The activity of purified LOX was stimulated by Mg2+ and Ca2+. 相似文献
11.
Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii 总被引:1,自引:0,他引:1
R. S. Ronimus Jurre Koning H. W. Morgan 《Extremophiles : life under extreme conditions》1999,3(2):121-129
The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg−1. The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH
optimum of 6.4), and the apparent K
m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V
max of 243 U mg−1) and 1.47 mM (apparent V
max of 197 U mg−1), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent
cations including Na+ and K+. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal
reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K
m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V
max of 2.9 U mg−1) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared
to ATP and pyrophosphate-dependent phosphofructokinases.
Received: August 14, 1998 / Accepted: December 2, 1998 相似文献
12.
A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag+ and Hg2+. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a Km of 3.09 mM and a Vmax of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a Km of 1.65 mM and a Vmax of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with Ki values of 0.033 and 468 mM, respectively. 相似文献
13.
Yukio Kimura Noriko Yasuda Hiroko Tanigaki-Nagae Toshikatsu Nakabayashi Hisami Matsunaga Masako Kimura 《Bioscience, biotechnology, and biochemistry》2013,77(10):2509-2516
Acetoacetate decarboxylase is a valuable tool for clinical analysis of ketone bodies in human plasma. After screening many microorganisms, we found Bacillus polymyxa A-57 to be a new enzyme source with a good yield of acetoacetate decarboxylase. After purifying the intracellular enzyme by three-stage column chromatography, we identified it as a single protein by SDS-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of approximately 280,000 and consisted of 10 (±2) identical subunits. It had an optimum pH of 5.9 and was stable up to about 60°C for 30min. The apparent Km and Vmax values for lithium acetoacetate were 0.94 mm and 296 μmiol/min/mg, respectively. In addition, the decarboxylase activity was found in the broth. After purification, we found that it was due to an active peptide which we named A-57-9, which we identified as the antibiotic polymyxin M1. However, the Vmax value (0.25 μmol/min/mg) of the peptide was very much lower and the Km value (400 mm) was higher than those of real acetoacetate decarboxylase. 相似文献
14.
Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K
m of acetate uptake was 50 μM and the V
max 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by
propionate with a K
i of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane
potential, but independent of the presence of Na+-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism. 相似文献
15.
Maja Milo?evi? Snje?ana Petrovi? Nata?a Veli?kovi? Ivana Grkovi? Marija Ignjatovi? Anica Horvat 《Molecular and cellular biochemistry》2012,371(1-2):199-208
Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K m values were 506.4?±?62.1 and 638.8?±?31.3?μM, with a calculated V max (app) of 3,973.0?±?279.5 and 2,853.9?±?79.8?nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5?mM). According to similar apparent Km values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology. 相似文献
16.
Wilfred Niels Arnold 《Current microbiology》1979,2(2):109-112
Torulopsis glabrata cells have a specific mechanism for endogenous trehalose mobilization that is associated with the lag phase attending new
growth. The degree of internal trehalose depletion depends upon the initial concentration of glucose in the medium. Exogenous
trehalose was shown to be assimilated via extra-cytoplasmic hydrolysis prior to, transport of the generated glucose into the
cytoplasm. The effect on internal trehalose depletion in this case was commensurate with slower presentation of free glucose
and was not indicative of the initial concentration of exterrial glucose residues as trehalose. Kinetic parameters for glucose
uptake wereK
m
=0.49 mM,V
max
=109 μmol/min/g dry weight; those for trehalose assimilation wereK
m
=2.5 mM,V
max
=17.4μmol/min/g dry weight. 相似文献
17.
Jan van Brederode Jean Chopin John Kamsteeg Gerrit van Nigtevecht Ria Heinsbroek 《Phytochemistry》1979,18(4):655-656
In Silene alba plants the dominant allele of gene Fg controls an enzyme which catalyses the formation of isovitexin 2″-O-glucoside both in petals and green parts. Both isovitexin and isoorientin can act as substrate. Kmvalues for the isovitexin glucosylation are 0.09 mM for isovitexin and 0.3 mM for UDP-glucose, Vmax 0.17 nmol min?1 mg protein?1. For the isoorientin glucosylation Km values of 0.45 mM for isoorientin, of 0.75 mM for UDP-glucose and Vmax of 0.27 nmol min?1 mg protein?1 are found. The pH optima for both substrates differ markedly. For the substrate with one hydroxyl in the B-ring, isovitexin, the pH optimum is pH 8.5. For isoorientin, which has two hydroxyls in the B-ring, a pH optimum of 7.5 is found. These results suggest that the B-ring hydroxylation pattern influences the pH at which the substrate has optimal affinity for the enzyme. The location of the carbon-carbon bound glucose on a the flavonoid skeleton is of importance for enzyme activity as well. Vitexin, which has glucose at the 8-position, was not a substrate. The glucosylation of vitexin could, however, be demonstrated in enzyme extracts of petals of plants, grown from seed collected in Armenia; in these petals apart from isovitexin glycosides, vitexin glycosides are found as well. 相似文献
18.
Cloning,expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli
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Curtis A. Lajoie Joshua B. Kitner Stephen J. Potochnik Jakob M. Townsend Christopher C. Beatty Christine J. Kelly 《Biotechnology progress》2016,32(5):1230-1237
Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a Vmax of 0.142 U/mg purified rXI, and a KM for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol KI values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low KM of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca2+ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher Vmax at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016 相似文献
19.
I. Michalík 《Biologia Plantarum》1982,24(3):161-169
In an experiment with native maize roots depending on different phosphorus concentration in the external solution (0.001 …
50 mM P), the multiphasic character of the kinetics of phosphate uptake has been stated. The single phases are characterized
by the different values of Km and Vmax. In the wide range of concentrations the isotherm of the phosphate uptake has five evident phases. The character of kinetics
for the uptake of phosphate is analogical to the kinetics of the enzymatic reactions described by the Michaelis-Menten equation.
On the other hand the linear dependence for the inactivated root was determined,i.e. the uptake of phosphate versus different phosphorus concentration in the external solution.
The graphic representation of the logarithmic values for the phosphorus taken up versus the different phosphorus concentration
in the external solution gives the biphasic course including concentration less than 1.0 mM P and more than 1.0 mM P. Within
the framework of the concentration range the following values of Vmax, Km and ϕin were calculated under the conditions if the concentration of phosphorus is less than 1.0mMP: Vmax = 1.705 μmol P × g-1h-1, Km = 0.057 mM P and ϕin = 0.83,i.e. if the concentration of phosphorus is more than 1.0mM P: Vmax = 40 μmol P × g-1 h-1, Km = 16.66 mM and ϕin = 20.
According to these results, the phosphate concentration in the external solution influences the activity of the transport
mechanisms concerning their conformative changes which discretely change their working regime of membrane transport. This
is also demonstrated in the change of values Vmax, Km and ϕin. 相似文献
20.
AbstractPurified Acetobacter tropicalis dextransucrase was immobilized in different matrices viz. calcium-alginate, κ-carrageenan, agar, agarose and polyacrylamide. Calcium-alginate was proved to be superior to the other matrices for immobilization of dextransucrase enzyme. Standardization of immobilization conditions in calcium-alginate resulted in 99.5% relative activity of dextransucrase. This is the first report with such a large amount of relative activity as compared to the previous reports. The immobilized enzyme retained activity for 11 batch reactions without a decrease in activity which suggested that enzyme can be used repetitively for 11 cycles. The dextransucrase was also characterized, which revealed that enzyme worked best at pH 5.5 and 37?°C for 30?min in both the free as well as immobilized state. Calcium-alginate immobilized dextransucrase of A. tropicalis showed the Km and Vmax values of 29?mM and 5000?U/mg, respectively. Free and immobilized enzyme produced 5.7?mg/mL and 2.6?mg/mL of dextran in 2?L bench scale fermenter under optimum reaction conditions. This immobilization method is very unconventional for purified large molecular weight dextran-free dextransucrase of A. tropicalis as this method is used usually for cells. Such reports on entrapment of purified enzyme are rarely documented. 相似文献