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1.
Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.
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Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin. 相似文献
2.
A. A. Raevskaya E. M. Kuznetsova M. V. Savvateeva S. E. Severin 《Biochemistry. Biokhimii?a》2010,75(7):866-872
A recombinant hepsin-producing strain of Escherichia coli was obtained and the conditions for hepsin expression in a bacterial system were optimized. To study the physicochemical
properties of the enzyme, a procedure for purification of active recombinant hepsin using metal-chelate affinity chromatography
and ion-exchange chromatography was developed. The interaction of recombinant hepsin with various peptide substrates is characterized.
The dose-dependent inhibition of the recombinant hepsin enzyme activity by anthralin in vitro and an increase in the hepsin enzymatic activity in the presence of resveratrol were revealed. 相似文献
3.
Oxaloacetate decarboxylase (OXAD), the enzyme that catalyzes the decarboxylation of oxaloacetate to pyruvic acid and carbon dioxide, was purified 245-fold to homogeneity from Pseudomonas stutzeri. The three-step purification procedure comprised anion-exchange chromatography, metal-chelate affinity chromatography, and biomimetic-dye affinity chromatography. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native high-performance gel-filtration liquid chromatography were, respectively, 63 and 64 kDa, suggesting a monomeric protein. OXAD required for maximum activity divalent metal cations such as Mn2+ and Mg2+ but not monovalent cations. The enzyme is not inhibited by avidin, but is competitively inhibited by adenosine 5'-diphosphate, acetic acid, phosphoenolpyruvate, malic acid, and oxalic acid. Initial velocity, product inhibition, and dead-end inhibition studies suggested a rapid-equilibrium ordered kinetic mechanism with Mn2+ being added to the enzyme first followed by oxaloacetate, and carbon dioxide is released first followed by pyruvate. Inhibition data as well as pH-dependence profiles and kinetic parameters are reported and discussed in terms of the mechanism operating for oxaloacetate decarboxylation. 相似文献
4.
Sequential isolation of superoxide dismutase and ajmaline from a tissue culture of Rauwolfia serpentina Benth 总被引:1,自引:0,他引:1
Two biologically active compounds, the enzyme superoxide dismutase (EC 1.15.1.1) and the antiarrhythmic indole alkaloid ajmaline, were isolated from a callus culture of Rauwolfia serpentina Benth. Sequential isolation of biologically active compounds by metal-chelate affinity chromatography followed by azoadsorbent affinity chromatography allowed us to obtain highly purified products. The yields of superoxide dismutase and ajmaline were 180 mg/kg biomass and 16.5 g/kg dry weight, respectively. 相似文献
5.
Glycogen synthase I was purified from bovine polymorphonuclear leucocytes (PMNs) by a procedure involving concanavalin A-Sepharose affinity chromatography. The purified glycogen-bound glycogen synthase I had a specific activity of 9.83 U/mg protein and the glycogen free enzyme 21 U/mg protein. Molecular ratio of the native enzyme and the subunit were 340 K and 85 K respectively. After phosphorylation by the catalytic subunit of cAMP-dependent protein kinase the phosphorylated sites were studied using high-performance liquid chromatography (HPLC) of the tryptic 32P-peptides. The enzyme was phosphorylated at three different sites with retention times identical to site 1a, site 1b, and site 2 from rabbit skeletal muscle glycogen synthase. 相似文献
6.
Aaron L. Berkovsky Pavel P. Potapov 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,692(2):273
Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH. 相似文献
7.
Andujar-Sánchez M Cámara-Artigas A Jara-Pérez V 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,783(1):247-252
Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site. 相似文献
8.
Properties of substrate-binding domains, some parameters of affinity sorbents, and a number of other special features that were necessary to take into account during creation of chromatographic system for isolation and purification of proteins with incorporated chitin-binding domain were discussed in this review. This method was shown to be successfully used along with metal-chelate affinity chromatography. The metal-chelate affinity chromatography with the use of polyhistidine peptides as affinity labels is successfully applied to isolation, purification, and investigation of recombinant proteins. However, this system had some disadvantages. At present, scientists attracted more and more attention to substrate-binding domains, including those chitin-binding, because they had a number of advantages being used as affinity label. 相似文献
9.
A Robichon 《Journal of cellular biochemistry》1991,47(2):147-157
The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate a specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion-exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a "pseudo-site" when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 mumol/min/mg. Moreover, this enzyme was phosphorylated by cAMP- and cGMP-dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases. 相似文献
10.
Hidalgo A Betancor L Mateo C Lopez-Gallego F Moreno R Berenguer J Guisán JM Fernández-Lafuente R 《Biotechnology progress》2004,20(5):1578-1582
A hexameric Mn-catalase was purified from crude extracts of Thermus thermophilus using ammonium sulfate precipitation and ion metal-chelate affinity chromatography (IMAC). Eupergit 250 and Sepabeads FP-EP3 epoxy supports derivatized with iminodiacetic acid (IDA) and copper were used, at similar micromole/packed milliliter of support. Although Eupergit 250-IDA-Cu support adsorbed 80% of the total proteins in the extract, it exhibited a minimum affinity for the catalase. On the other hand, Sepabeads FP-EP3-IDA-Cu allowed the full adsorption of the catalase activity, which could be desorbed in fractions of different purity. This was attributed to a different geometrical congruence of the support surfaces with the enzyme surface, resulting in a different ability to form multipoint interactions with the proteins. Thus, by a cleanup step, followed by a negative chromatographic step using Eupergit 250-IDA-Cu2+ and by the adsorption of the catalase on Sepabeads-IDA-Cu2+ support, a pure enzyme fraction was obtained and its N-terminal end was sequenced. 相似文献
11.
《Bioscience, biotechnology, and biochemistry》2013,77(1):256-260
Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin. 相似文献
12.
Type II hexokinase (EC 2.7.1.1) has been purified from rat skeletal muscle by a simple procedure involving chromatography on DEAE-cellulose, affinity elution chromatography from phosphocellulose, and gel filtration on Sephadex G-200. The key to the preparation of homogeneous enzyme is the affinity elution step in which an effector molecule, glucose 6-phosphate, is used as the eluting ligand. A 5300-fold purification is obtained by the procedure and over 400-fold purification is obtained in the affinity elution step alone. Approximately 3.3 mg of homogeneous hexokinase with a specific activity of 120 units/mg is obtained from 800 g of rat limb. 相似文献
13.
Puri M Kaur A Schwarz WH Singh S Kennedy JF 《International journal of biological macromolecules》2011,48(1):58-62
Kinnow peel, a waste rich in glycosylated phenolic substances, is the principal by-product of the citrus fruit processing industry and its disposal is becoming a major problem. This peel is rich in naringin and may be used for rhamnose production by utilizing α-L-rhamnosidase (EC 3.2.1.40), an enzyme that catalyzes the cleavage of terminal rhamnosyl groups from naringin to yield prunin and rhamnose. In this work, infrared (IR) spectroscopy confirmed molecular characteristics of naringin extracted from kinnow peel waste. Further, recombinant α-L-rhamnosidase purified from Escherichia coli cells using immobilized metal-chelate affinity chromatography (IMAC) was used for naringin hydrolysis. The purified enzyme was inhibited by Hg2+ (1 mM), 4-hydroxymercuribenzoate (0.1 mM) and cyanamide (0.1 mM). The purified enzyme established hydrolysis of naringin extracted from kinnow peel and thus endorses its industrial applicability for producing rhamnose. 相似文献
14.
Nakagawa T Nishiuchi K Akaki J Iwata H Satou R Suzuki F Nakamura Y 《Bioscience, biotechnology, and biochemistry》2007,71(1):256-260
Human prorenin attached by a decahistidine tag at the C-terminus was produced in Chinese hamster ovary cells. The tagged protein secreted into the culture medium was in the inactive prorenin form, and was activated to mature renin by proteolytic removal of its prosegment by trypsin in the same manner as native prorenin. The tagged (pro)renin was efficiently purified by metal-chelate affinity chromatography. The enzymatic properties of mature renin carrying the tag were similar to native renin. These results indicate that the introduction of a decahistidine tag at the C-terminus does not interfere with either the correct folding of prorenin or the catalytic activity of mature renin. 相似文献
15.
Purification of human cytidine deaminase: molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs 总被引:2,自引:0,他引:2
T Cacciamani A Vita G Cristalli S Vincenzetti P Natalini S Ruggieri A Amici G Magni 《Archives of biochemistry and biophysics》1991,290(2):285-292
Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting. 相似文献
16.
The effect of proteolysis on the calmodulin activation of cyclic nucleotide phosphodiesterase 总被引:5,自引:0,他引:5
A high Km cytoplasmic cyclic nucleotide phosphodiesterase (EC 3.4.1.17) has been obtained from bovine brain. The unproteolyzed enzyme contains (63 +/- 1) X 10(3) molecular weight polypeptide chains which exhibit little if any basal catalytic activity. Complexation with calmodulin stimulates the catalytic activity nearly 2 orders of magnitude, presumably, by causing a conformational change in the enzyme which either creates or exposes the catalytic sites. Removal of about 120 amino acids from the terminal portion(s) of each polypeptide chain either by an endogenous protease or by exogenous trypsin prevents calmodulin complexation and generates a basal catalytic activity equivalent to that of the unproteolyzed enzyme-calmodulin complex. In contrast to affinity chromatography using immobilized calmodulin, blue dextran-Sepharose chromatography can be used to select for enzyme containing only unproteolyzed polypeptide chains. 相似文献
17.
Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase 总被引:1,自引:0,他引:1
A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme. 相似文献
18.
In order to facilitate the purification of 1,2-alpha-mannosidase from an enzyme product of Aspergillus oryzae, we have devised a rapid and simple procedure. A partially purified enzyme preparation obtained from the A. oryzae enzyme product, by means of ammonium sulfate fractionation followed by CM-Sephadex C-50 chromatography, was subjected to affinity chromatography with baker's yeast mannan gel as an adsorbent. 1,2-alpha-Mannosidase was retarded and well separated from the major protein peak on the affinity column. After a second affinity chromatography under the same conditions, 1,2-alpha-mannosidase was finally purified 7,500-fold with a 22.9% yield. The enzyme preparation thus obtained was quite suitable for the structural analysis of glycoconjugates. 相似文献
19.
The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5-phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal-5-P per dimer, three forms of the enzyme may exist in solution: apoenzyme-2 pyridoxal 5'-phosphate, apoenzyme-1 pyridoxal 5'-phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme-2 pyridoxal 5'-phosphate/apoenzyme-1 pytidoxal 5'-phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal-5-P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme-1 pyridoxal 5'-phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4-pyridoxic-5-P (KD equals 1.1 times 10- minus 6 M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by two independent methods, lend strong support to the hypothesis that the two subunits of the enzyme function independently. 相似文献
20.
The purification and properties of a beta-N-acetylhexosaminidase from Trichomonas foetus. 总被引:1,自引:0,他引:1
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A beta-N-acetylhexosaminidase was purified 800-fold from extracts of Trichomonas foetus by affinity chromatography on a column of N-(epsilon-aminohexanoyl)-2-acetamido-2-deoxy-beta-D-glucopyranosylamine bound to CNBr-activated Sepharose. The enzyme has a dual specificity for the p-nitrophenyl beta-D-glycosides of N-acetylglucosamine and N-acetyl-galactosamine. The parent sugars are both competitive inhibitors. The enzyme has a mol. wt. approx. 150000 and a pH optimum of 6.2. It is suggested that the same active site catalyses both activities and that no part is played by the 4-hydroxyl group in substrate binding, but it is involved in determining the catalytic rate. 相似文献