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1.
Cytosolic phosphofructokinase from spinach leaves : I. Purification, characteristics, and regulation
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Cytosolic ATP-dependent phosphofructokinase (PFK) from spinach leaves (Spinacia oleracea L.) was enriched 2600-fold by (NH4)2SO4 fractionation, DEAE anion exchange chromatography, Blue Sepharose CL-6B, and ATP agarose type 3-affinity chromatography. The final preparation had a specific activity of 417 nkat per milligram protein and exhibited four bands between 50 and 70 kilodaltons following denaturing electrophoresis. Only one band of ATP- and fructose 6-phosphate (F-6-P)-dependent, Pistimulated activity was detected following isoelectric focusing PAGE and nondenaturing discontinuous PAGE of the final preparation. Crude extracts contained, in addition to the band observed in the final preparation, a second band that was inhibited by Pi. The latter band is presumably chloroplastic PFK. PFK was stimulated by the anions Pi2−, Cl−, SO42−, NO3−, HAsO42−, and HCO3− but was not affected by NH4+. Pi and Mg2+ changed the response of PFK toward pH and affected the saturation kinetics of F-6-P. In general, activity was highest when Pi was high and (or) Mg2+ was low. Phosphoenolpyruvate (PEP), 2-PGA, and PPi, but not 3-PGA, inhibited PFK. Although the inhibition by PEP and 2-PGA was reduced or relieved by Pi, the inhibition by PPi was not affected by Pi. F-2, 6-P2 had no effect upon the activity of PFK. It is proposed that, in the cytosol of spinach leaves, PFK is likely to be more active during the dark, when cytosolic Pi levels are high, than in the light. 相似文献
2.
Peter Traub 《Archives of biochemistry and biophysics》1984,228(1):120-132
The Ca2+-activated, neutral thiol proteinase specific for intermediate filament subunit proteins was isolated at large scale from the postribosomal supernatant of a low-ionic-strength extract of porcine kidney and Ehrlich ascites tumor (EAT) cells, respectively. The purification procedure encompassed DEAE-Sephacel ion exchange chromatography of the material precipitating between 23 and 55% (NH4)2SO4 saturation, followed by hyroxylapatite chromatography and activated thiol Sepharose 4B covalent chromatography. On the average, 25 mg of 62% pure enzyme was obtained from 500 g frozen kidney and 55 mg of 51% pure enzyme from 500 g EAT cells within a week. Both enzyme preparations were free of Ca2+-independent proteolytic activities and indistinguishable with respect to their physicochemical and functional properties; their catalytic properties were indistinguishable from those of enzyme purified to homogeneity on arginine methylester Sepharose 4B. Because of this identity, porcine kidney proves to be an inexpensive source for the Ca2+-activated proteinase which had previously been isolated and purified at small scale from EAT cells (W. J. Nelson and P. Traub, (1983) J. Biol. Chem.257, 5544–5553). Despite a 38% protein contamination, the partially purified enzyme from porcine kidney is useful for the in vitro study of structure-function relationships of intermediate filaments and their subunit proteins. During affinity chromatography of the partially purified proteinase from EAT cells on arginine methylester Sepharose 4B, a 100-kDa protein was purified which has a high affinity for arginine residues. It also occurs in porcine kidney, although at a considerably lower concentration. Its cellular localization and function remain to be determined. 相似文献
3.
Two S-adenosylmethionine-dependent indolethylamine N-methyltransferase activities were purified from soluble extracts of Phalaris tuberosa by fractionation with (NH4)2SO4, and chromatography on DEAE-Sephadex. One enzyme methylated the primary indolethylamines and the other methylated the secondary indolethylamines in the plant. These two enzymes were similar in catalytic and bulk physical properties and could not be separated during purification or by A 1.5 Agarose or hydroxylapatite chromatography. The products of enzymic reactions were identified by TLC. 相似文献
4.
《Process Biochemistry》2010,45(7):1172-1175
Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t-butanol, the (NH4)2SO4 concentration and the TPP cycles. The ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 20% (NH4)2SO4 resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH4)2SO4 to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels. 相似文献
5.
J.E. Escamilla M. Contreras A. Martínez M. Zentella-Piña 《Archives of biochemistry and biophysics》1982,218(1):275-285
The purification procedure of milligram quantities of stable myoinositol-1-phosphate synthase (EC 5.5.1.4) from Neurospora crassa is reported. The procedure includes: (a) (NH4)2SO4 and protamine sulfate precipitations, (b) gel filtration in Ultrogel AcA-34 (LKB), (c) DEAE-cellulose chromatography, (d) AH-Sepharose 4B chromatography, and (e) calcium phosphate gel chromatography. The enzyme is considered pure according to the following criteria: (a) gel filtration, (b) sucrose density gradient centrifugation, (c) polyacrylamide gel electrophoresis, and (d) isoelectric focusing technique. The molecular weight estimated by gel filtration chromatography and sucrose density gradient centrifugation is 345,000. The subunit molecular weight is 59,000. The active enzyme seems to posses an hexameric structure. The isoelectric point estimated for the pure enzyme is 5.2. The enzyme was optimally stimulated by 10 mm (NH4)2SO4 and by 50 mm KCl, while NaCl had a minor inhibitory effect at higher concentrations. The divalent cations Mg2+ and Mn2+ were inhibitory only at nonphysiological concentrations. The enzymatic activity after the salt fractionation steps was about 33% NAD+ independent; but with purification the resulting homogeneous enzyme showed less than 5% NAD+-independent activity. 相似文献
6.
A simple method for the purification of the soluble oligomycin-insensitive mitochondrial ATPase from heart is described. It consists of adsorption of the of the enzyme to Sepharose hexylammonium followed by elution with KCl and a precipitation step with (NH4)2SO4. In sodium dodec'yl sulfate gels, the enzyme shows the A, B, C, D, and E subunits; however, the D and E subunits appear only when the gels are loaded with a high concentration of protein. The Km for Mg-ATP is approximately 0.6 mm and is inhibited by ADP. 相似文献
7.
Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.
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Rat fibrinogen was purified from rat plasma by using lysine–Sepharose chromatography, repeated precipitation with 25%-satd. (NH4)2SO4 and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70–75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aα-, Bβ- and γ-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem. 250, 4678–4683; Stemberger & Jilek (1976) Thromb. Res. 9, 657–660], in which no separation between Aα- and Bβ-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aα-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure. 相似文献
8.
Formation of delta-Aminolevulinic Acid from Glutamic Acid in Algal Extracts : Separation into an RNA and Three Required Enzyme Components by Serial Affinity Chromatography
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Extracts from plant chloroplasts and algae catalyze the conversion of glutamate to δ-aminolevulinic acid (ALA) in the first committed step of the tetrapyrrole biosynthetic pathway leading to chlorophylls, hemes, and bilins. The conversion requires ATP, Mg2+, and NADPH as cofactors. Soluble extracts from Chlorella vulgaris have now been resolved into four macromolecular fractions, all of which are required to reconstitute activity. One fraction contains a low molecular weight RNA which can be separated from the protein components in an active high-speed supernatant by treatment with 1 molar NaCl followed by precipitation of the proteins with (NH4)2SO4 at 70% saturation. The proteins recovered from the (NH4)2SO4 precipitate are reactivated by addition of a fraction containing tRNAs isolated from Chlorella by phenol-chloroform extraction and DEAE cellulose chromatography. Three required protein fractions were resolved from the RNA-depleted (NH4)2SO4 precipitate by serial affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. Glycerol was found to stabilize the enzyme activity during the separation process. The majority of the glutamate:tRNA ligase activity was associated with the fraction which was retained by Blue-Sepharose and not retained by ADP-agarose, in agreement with the reported properties of the affinity ligands. The active material in the fraction not retained by Blue-Sepharose eluted as a single component on gel filtration chromatography, with an apparent molecular weight of 67,000. The active component in the RNA fraction also eluted as a single component on gel filtration chromatography. 相似文献
9.
Hydrophobic zeolite Y can be used as a fast and efficient and inexpensive matrix in the purification of proteins from crude extracts. Preferably the zeolite can be used in the first purification step, replacing the commonly used precipitation techniques with (NH4)2SO4 or ethanol. The time required for the zeolite prefractionation was a few hours compared to the much more time consuming precipitation procedure which demands centrifugation and subsequent dialysis. Proteins can be adsorbed on the zeolite either in order to remove undesired proteins or to be subsequently eluted from the zeolite in order to achieve purification and concentration. Removal of undesired proteins is exemplified by the purification of horseradish peroxidase from a crude extract. The zeolite procedure enhanced the specific activity five times and provided a yield similar to that which was obtained by the use of standard procedures, (NH4)2SO4 fractionation and ion-exchange chromatography. Binding and subsequent elution of proteins from the zeolite is exemplified by the purification of monoclonal antibodies from hybridoma culture supernatants. Proteins were desorbed from the zeolite by the use of polyethylene glycol 600 and this procedure yielded a purification factor of 5. 相似文献
10.
A rapid and simple method for the purification of rat liver alkaline RNase inhibitor from a 105,000 g supernatant is reported. It involves protein precipitations by (NH4)2SO4 and chromatography on carboxymethyl cellulose-RNase column. The purification procedure gives a 1020-fold increase in specific activity with a yield of 32%. This purified inhibitor can be stored for 5 weeks without any loss in activity. 相似文献
11.
Gel filtration chromatography with Sepharose agarose gel has been widely applied in the purification of enzymes because of its capability to separate macromolecules according to molecular size. Although a wide range of pH and salt concentrations have been suggested for its use, we have found that the selectivity, or efficiency, of separation is strongly affected by the pH and salt concentrations actually used. Separation is best at neutral pH with low salt concentrations. Increasing the molarity of the buffer or salt content (such as ammonium sulfate) in the protein sample will either broaden protein peaks resulting in poor separation or displace the peaks to a position of much lower apparent hydrodynamic volume. Rabbit plasma monoamine oxidase (MAO), a protein of 150,000 MW, when combined with 1.3 m (NH4)2SO4 at pH 5.4, was found to be retained in Sepharose 6B column until the very end and elute with ammonium sulfate molecules. This behavior was attributed to severe morphological changes on the gel surface at acidic pH leading to a loss of selectivity. Evidence for this interpretation is provided by parallel experiments with Sephadex columns under identical conditions which excludes the possibility of dissociation of MAO into subunits and by scanning electron microscopy which demonstrates the change of surface morphology of the gel. The necesslty of a careful selection of optimum conditions for Sepharose gel chromatographic separation is therefore suggested. 相似文献
12.
Barbara Egin-Bühler Rosemarie Loyal Jürgen Ebel 《Archives of biochemistry and biophysics》1980,203(1):90-100
The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley (Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH4)2SO4, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit (Mr = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component (Mr = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — Mr component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated. 相似文献
13.
The salt-soluble proteins of groundnut meal were fractionated by precipitation with (NH4)2SO4 by increasing the (NH4)2SO4 saturation in steps of 10%. The sharp separation into arachin and conarachin claimed by earlier workers was not achieved, as protein was precipitated at each stage from 20 to 100% saturation with (NH4)2SO4. The fractions so obtained were examined by disc electrophoresis on polyacrylamide gel and the amino acid compositions were determined by ion-exchange chromatography. Differences in both electrophoretic pattern and amino acid composition were found. The protein precipitated by CaCl2 solution was similar in yield, nitrogen content, electrophoretic pattern, and amino acid composition to the fraction precipitating at 10–20% (NH4)2SO4 saturation. The main differences in amino acid composition of the various fractions precipitated by (NH4)2SO4 were found in the amino acids cystine, methionine, and lysine, which increased with increase in (NH4)2SO4 saturation. The electrophoretic pattern and amino acid composition of “conarachin” varied according to the method of preparation. 相似文献
14.
Qiudong Su Yao Yi Minzhuo Guo Feng Qiu Zhiyuan Jia Xuexin Lu Qingling Meng Shengli Bi 《Biochemical and biophysical research communications》2013
Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire ‘α’ antigenic determinant amino acids (aa) 119–152 of HBsAg into the truncated HBc (aa 1–144), between Asp78 and Pro79. Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH4)2SO4 precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire ‘α’ antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines. 相似文献
15.
Hyun-Ho Lee Sang-Joon Kim Hyun-Jae Shin Ji-Yeon Park Ji-Won Yang 《Biotechnology and Bioprocess Engineering》2002,7(6):375-379
Methyl mercaptan oxidase was successfully induced inThiobacillus thioparus TK-m using methyl mercaptan gas, and was purified for the detection of mercaptans. The purification procedure involved a
DEAE (diethylaminoethyl)-Sephacel, or Superose 12, column chromatography, with recovery yields of 47.5 and 48.5%, and specific
activities of 374 and 1240.8 units/mg-protein, respectively. The molecular weight of the purified methyl mercaptan oxidase
was 66.1kDa, as determined by SDS-PAGE. The extract, from gel filtration chromatography, oxidizes methyl mercaptan, producing
formaldehyde, which can be easily detected by the purpald-coloring method. The optimized temperature for activity was found
to be at 55°C. This enzyme was inhibited by both NH4Cl and (NH4)2SO4, but was unaffected by either KCl or NaCl at less than 200 mM. With K2SO4, the activity decreased at 20 mM, but recovered at 150 mM. In the presence of methanol, full activity was maintained, but
decreased in the presence of glycerin, ethanol and acetone 43, 78 and 75%, respectively. 相似文献
16.
Voltaire Sant’Anna Ana Paula Folmer Correa Amanda de Souza da Motta 《Preparative biochemistry & biotechnology》2016,46(8):838-843
Antimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH4)2SO4], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH4)2SO4 system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH4)2SO4 and 1.0?mol l?1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34. 相似文献
17.
《Process Biochemistry》2007,42(9):1296-1301
Recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH) partitioning was studied in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The objectives of this work were to investigate influences; varying the molecular mass and concentration of PEG, pH, phase volume ratio (VR), tie-line length (TLL) and concentration of (NH4)2SO4 on the partition behavior of PheDH. It was revealed that the partitioning was not affected by VR, while PEG molecular mass and concentration and (NH4)2SO4 concentration had significant effects on enzyme partitioning. Longer TLL and higher pH resulted in better partitioning into the top phase. Under the most favorable partition conditions with 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH4)2SO4 and VR = 0.25 at pH 8.0, partition coefficient (KE), recovery (R%), yield (Y%) and TLL were achieved 58.7%, 135%, 94.42% and 39.89% (w/w), respectively. Overall, the promising results obtained in this research indicated that the ATPS partitioning can be provided an efficient and powerful tool for recovery and purification of recombinant PheDH. 相似文献
18.
Evidence for the presence in tobacco leaves of multiple enzymes for the oxidation of glycolate and glyoxylate 总被引:7,自引:2,他引:5
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Havir EA 《Plant physiology》1983,71(4):874-878
The enzymic oxidation of glycolate to glyoxylate and glyoxylate to oxalate by preparations purified from tobacco (Nicotiana tabacum var Havana Seed) leaves was studied. The Km values for glycolate and glyoxylate were 0.26 and 1.0 millimolar, respectively. The ratio of glycolate to glyoxylate oxidation was 3 to 4 in crude extracts but decreased to 1.2 to 1.5 on purification by (NH4)2SO4 fractionation and chromatography on agarose A-15 and hydroxylapatite. This level of glyoxylate oxidation activity was higher than that previously found for glycolate oxidase (EC 1.1.3.1). The ratio of the two activities was changed by reaction with the substrate analog 2-hydroxy-3-butynoate (HBA) which at all concentrations inhibited glyoxylate oxidation to a greater extent than glycolate oxidation. The ratio of the two activities could also be altered by changing the O2 concentration. Glycolate oxidation increased 3.6-fold when the O2 atmosphere was increased from 21 to 100%, whereas glyoxylate oxidation increased only 1.6-fold under the same conditions. These changes in ratio during purification, on inhibition by HBA, and under varying O2 concentrations imply that tobacco leaves contain at least two enzymes capable of oxidizing glycolate and glyoxylate. 相似文献
19.
《Environmental Pollution (1970)》1978,15(3):235-240
Oil degradation was determined in oil-polluted (1 or 2 ml of light Nigerian crude/20 g soil, equivalent to 5 and 10% pollution) soils treated with (NH4)2SO4 and with nutrient elements with and without enhanced aeration. There was no significant difference in oil degradation in soils with and without enhanced aeration, nor in soils treated with and without (NH4)2SO4 and/or nutrients after 4 weeks incubation. After 12 weeks, oil degradation was significantly higher (p = 0·05) in the (NH4)2SO4 and nutrient treated soils in comparison to the untreated soils, and in soils with enhanced aeration in comparison to the undisturbed soil, at the 5% oil pollution level.Warburg respirometer studies showed more oxygen consumption (significant at p = 0·05) in the polluted soils compared with the unpolluted soils. In oil-polluted soils oxygen consumption was depressed significantly (p = 0·05) by the addition of (NH4)2SO4, but was enhanced significantly (p = 0·05) by the addition of (NH4)2SO4 and nutrient elements. The respiratory quotient (RQ) was reduced from 0·81 in unpolluted soils to 0·62 in oil-polluted soils. 相似文献
20.
Tadao Kamikawa Fujito Higuchi Makoto Taniguchi Yukihiro Asaka 《Bioscience, biotechnology, and biochemistry》2013,77(3):691-692
Two ribonucleases (RNases) designated RNase I and RNase II were found in Euphausia superba and isolated by (NH4)2SO4 fractionation, 2 cycles of CM-cellulose chromatography and gel filtration on Sephadex G-100. This procedure resulted in a 2,116-fold purification of RNase I and a 130-fold purification of RNase II. The molecular weight of both purified enzymes was estimated by gel filtration to be 31,500. The isoelectric points were 6.0 (RNase I) and 7.0 (RNase II). Each enzyme hydrolyzed poly A-U, poly U but did not degrade poly G, poly C and DNA. Both enzymes were classified as endonuclease from the hydrolysis product of yeast RNA and poly A. The enzymes were located mainly in the cardiac and pyloric portion of the stomach. 相似文献