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1.
Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide. 相似文献
2.
Summary An enzyme treatment and ammonium sulfate precipitation procedure was adapted to the detection of staphylococcal enterotoxin from high-protein foods. The enterotoxin is extracted from food with distilled water, after which soluble proteins are acid-precipitated (pH 4.5) and the supernatant washed with chloroform (pH 7.5). The extract is then treated with trypsin andPseudomonas peptidase for 2 h at +37°C. Residual unhydrolyzed material is precipitated with 60% ammonium sulfate for 15 min at +4°C. The precipitate is redissolved in phosphate buffer and concentrated by dialysis against polyethyleneglycol. The concentrate is washed with chloroform and lyophilized. The dry material is dissolved in 0.2 ml distilled water and enterotoxin detected by the micro-slide method with 24 h incubation at +37°C. Using this method, it has been possible within three days to detect 0.2–1.0 g staphylococcal enterotoxin A added to minced meat, dry sausage, smoked fish, cheese and milk. 相似文献
3.
Globulins were prepared by repeated precipitation with 1.3 M (NH4)2SO4 from a 0.7 M NaCl extract of milled rice. Isoelectric precipitation at pH 4.5 did not effectively remove the α-globulin from the others. A major fraction that remained in solution during dialysis of the globulin precipitate against water was similar in properties to the globulin soluble at pH 4.5 during the isoelectric precipitation process. Some properties of this water-soluble globulin fraction are reported. Proteins extracted from milled rice at 50° with 0.5 M NaCl and precipitated as 1- to 3-μm particles on cooling were verified to be globulins. 相似文献
4.
An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid. 相似文献
5.
An acidic protein was extracted with neutral-salt solutions from rat skin. When precipitated by dialysis against dilute acetic acid, the structural protein was separated from contaminating soluble collagens and other soluble proteins. The precipitate was dissolved in buffers containing 1% Triton X-100 and purified to apparent size and charge homogeneity by chromatography on a DEAE Bio-Gel A column. Triton X-100 was necessary for achieving nondestructive disaggregation of the protein which could be reversed by dialyzing out the detergent against methanol-dilute acetic acid. 相似文献
6.
Reassembly of a fimbrial hemagglutinin from Pseudomonas solanacearum after purification of the subunit by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 总被引:2,自引:1,他引:1
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Distilled water homogenates of Pseudomonas solanacearum B1, a highly fimbriated strain, strongly agglutinated human group A erythrocytes. The fimbriae and hemagglutinating activity were precipitated from the crude extract with 1% acetic acid, redissolved at pH 10, and precipitated again with 20 mM CaCl2 at pH 6.9. Ca2+, Mg2+, and Zn2+ had similar ability to precipitate the fimbrial hemagglutinin, but Na+ and K+ were much less effective. The fimbrial protein in the precipitate was purified to homogeneity by preparative gel electrophoresis in sodium dodecyl sulfate. The major protein band was eluted, and sodium dodecyl sulfate was removed by chromatography on ion retardation resin (AG 11A8) in 6 M urea. After dialysis against 10 mM sodium acetate (pH 4.5) to remove the urea, the protein reassembled to yield long fibers. These fibers were identical to fimbriae in the crude extract in diameter (6 nm) and in their ability to cause hemagglutination. The purified fimbriae contained no carbohydrates and wee similar to other bacterial fimbriae in amino acid composition, with hydrophobic amino acids comprising 41.8% of the total. 相似文献
7.
H Hayashi 《Biochimica et biophysica acta》1979,585(2):220-228
Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5--9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation. After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37 degrees C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process. From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes. 相似文献
8.
Membrane-free washed myofibrils derived from rat skeletal muscle homogenates contained a chymostatin-sensitive protease(s) which acted on associated myofibrillar proteins, at an optimum pH of 8.5, much less rapidly at low ionic strength (insoluble myofilaments) than at high salt concentrations (solubilized proteins). When the myofibrillar fraction was added to the particle-free cytosol prepared from the muscle extracts, proteins of the cytosol were also degraded, but the activity in this case was much more pronounced at low ionic strength. This was because inhibitor(s) of the proteinase present in the cytosol fraction were only effective at high ionic strength when all the myofibrillar (and associated) proteins were in solution. The protease was separated from the bulk of the myofibrillar proteins by gel chromatography at high ionic strength. On dialysis against a low-salt buffer, part of the enzyme was precipitated. The putative cytosolic inhibitor(s) were again only effective on the soluble enzyme at high ionic strength. 相似文献
9.
The solubility of palmitoyl-CoA is strongly affected by Mg2+ concentrations commonly used in acyltransferase reactions. In 0.10 M Tris-HCl buffer at pH 7.4 or 8.5, all of the palmitoyl-CoA in 10 microM solutions and 90% of the palmitoyl-CoA in 100 microM solutions are precipitated by 1 mM Mg2+. In 0.05 M phosphate at pH 7.4, and in 0.10 M Tris-HCl containing 0.4 M KCl, the substrate remains soluble at Mg2+ concentrations below 4-5 mM. Above 5 mM Mg2+, palmitoyl-CoA is insoluble in all of these buffers. Substrate solubility could therefore be a limiting factor when free Mg2+ and fatty acyl-CoAs are present together during acyltransferase assays. 相似文献
10.
Hindenori Hayashi 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,585(2):220-228
Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes. 相似文献
11.
Pyridoxaminephosphate oxidase (EC 1.4.3.5, deaminating) that was partially purified about 40-fold from dry baker's yeast was immobilized to iodo- and bromoacetyl polysaccharides. The most effective carrier was an iodoacetyl cellulose, to which almost complete activity of pyridoxine 5'-phosphate oxidase was immobilized in 0.02M potassium phosphate buffer (pH 8.5) containing 2M ammonium sulfate at 4 degrees C. The immobilized enzyme was more stable than the purified, soluble enzyme against heat and pH change. It was confirmed that N-(5'-phosphopyridoxyl)-L-serine was degradedly oxidized to pyridoxal 5'-phosphate and L-serine by the immobilized enzyme as comparable rate as pyridoxine 5'-phosphate, whereas N-(5'-phosphopyridoxyl)-D-serine did not serve as substrate, as in the purified, soluble enzyme. 相似文献
12.
目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。 相似文献
13.
Kim do Y Rha E Choi SL Song JJ Hong SP Sung MH Lee SG 《Journal of microbiology and biotechnology》2007,17(1):116-122
An efficient enzyme system for the synthesis of L-tyrosine was developed using a fed-batch reactor with continuous feeding of phenol, pyruvate, and ammonia. A thermo- and chemostable tyrosine phenol-lyase from Symbiobacterium toebii was employed as the biocatalyst in this work. The enzyme was produced using a constitutive expression system in Escherichia coli BL21, and prepared as a soluble extract by rapid clarification, involving treatment with 40% methanol in the presence of excess ammonium chloride. The stability of the enzyme was maintained for at least 18 h under the synthesis conditions, including 75 mM phenol at pH 8.5 and 40 degrees C. The fed-batch system (working volume, 0.5 1) containing 1.0 kU of the enzyme preparation was continuously fed with two substrate preparations: one containing 2.2 M phenol and 2.4 M sodium pyruvate, and the other containing 0.4 mM pyridoxal-5-phosphate and 4 M ammonium chloride (pH 8.5). The system produced 130 g/l of L-tyrosine within 30 h, mostly as precipitated particles, upon continuous feeding of the substrates for 22 h. The maximum conversion yield of L-tyrosine was 94% on the basis of the supplied phenol. 相似文献
14.
A method is described for the preparation of both Factor X1 and Factor X2 from citrated bovine blood. The proteins from the plasma were first adsorbed on barium citrate by adding barium chloride solution. The precipitate formed was stirred with citrate/NaOH pH 6.9 buffer; barium and other clotting factors were removed by adding ammonium sulphate (up to 30% saturation) to the suspension. The Factor X was then precipitated by 65% ammonium sulphate, after resolution in citrate buffer chromatographed on DEAE-Sephadex and purified by rechromatography on DEAE-Sephadex and DEAE-Sepharose, respectively. This yielded Factor X1 and Factor X2 with respective purifications of about 16 000 and 24 000-fold that of the plasma. The apparent molecular mass of both Factor X1 and Factor X2 was 55 kDa as estimated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Factor X2 had a higher specific biological activity of about 340 000 units/mg compared to that of Factor X1 of about 230 000 units/mg. 相似文献
15.
H S Brand G H Korver R J van de Stadt G P van Kampen J K van der Korst 《Biological chemistry Hoppe-Seyler》1990,371(7):581-587
Large proteoglycan monomers and small dermatan sulfate proteoglycans were extracted from explants of bovine articular cartilage with increasing (0-4 M) concentrations of guanidinium chloride (GuHCl). The first extractions were followed by a second extraction with 4 M GuHCl. The amount of proteoglycans extracted in the first buffer depended on the GuHCl concentration. At low concentrations of GuHCl, a relatively high amount of small proteoglycans was obtained. Fifty percent of the small proteoglycans was extracted in buffer with 0.85 M GuHCl, while 2.0-2.2 M GuHCl was needed to extract half of the large proteoglycans. Immediately after synthesis, 35S-labeled large proteoglycans were extracted much easier (50% at 1.4 M GuHCl), and those extracted at low concentrations of GuHCl were less capable of aggregation with hyaluronic acid. After 7 days of 'chase' these differences between endogenous and 35S-labeled proteoglycans had disappeared. 相似文献
16.
4-Oxalocrotonate tautomerase (4-OT) is a bacterial enzyme that is comprised of 6 identical 62 amino acid subunits. The 4-OT enzyme is an attractive model system in which to study the interrelationship between protein folding, subunit assembly, and catalytic function. Here we report on the GuHCl-induced equilibrium unfolding properties of wild-type 4-OT using catalytic activity measurements and using far-UV circular dichroism (CD) spectroscopy. We demonstrate that the unfolding of wild-type 4-OT in 50 mM phosphate buffers containing 6 M GuHCl is reversible at pHs 6.0, 7.4, and 8.5; and we find that there is both an enzyme concentration dependence and a pH dependence to the equilibrium unfolding properties of 4-OT. Our data suggests that the GuHCl-induced unfolding of 4-OT in 50 mM phosphate buffer at pH 8.5 can be modeled as a two-state process involving folded hexamer and unfolded monomer. On the basis of this model, we determined a free-energy value for the unfolding of 4-OT at pH 8.5 to be 68.7 +/- 3.2 kcal/mol under standard state conditions (1 M hexamer). In 50 mM phosphate buffers at pHs 6.0 and 7.4, only the catalytic activity denaturation curves are consistent with a two-state folding mechanism. At the lower pHs the far-UV-CD transitions are not well described by a two-state model. Our results at pHs 6.0 and 7.4 suggest that intermediate state(s) are populated in the equilibrium unfolding reaction at these lower pHs and that these intermediate state(s) have some helical content but no measurable catalytic activity. 相似文献
17.
A rapid procedure for the isolation of 3,4-dihydroxyphenylalanine-containing proteins has been developed in which the protein is selectively bound to a m-phenylboronate agarose column, and eluted with 1.0 M ammonium acetate, pH 3.0. The method is based on the affinity of boronates for diols including catechol. The chromatography is carried out in the absence of oxygen to prevent oxidation of the catechol. Other proteins are eluted beforehand with 0.25 M ammonium acetate, pH 8.5, or for glycoproteins with a Tris buffer containing 0.2 M sorbitol, pH 8.5. 相似文献
18.
Prekeratin was isolated from bovine snout epidermis with 0.1 M citric acid/sodium citrate buffer, pH 2.6 (buffer A). Filaments, 6.0-9.0 nm wide, were produced by dialysis against low ionic strength buffer A or by dissociating prekeratin in 8 M urea solution followed by dialysis against 0.005 M Tris-HCl buffer, pH 8.0. The polypeptide composition of both prekeratin and filaments was studied by four different SDS-polyacrylamide gel electrophoresis methods. The best resolution was obtained by Laemmli's technique in which both prekeratin and filaments were separated into three major and seven distinct minor bands of polypeptides. The major ones comprise approx. 70% of total polypeptides and their estimated molecular weights are 68 000, 54 000, and 50 000. The molecular weight of minor ones is in decreasing order 65 000, 63 000, 61 000, 58 000, 47 000, 44 000 and 42 000. It is proposed that the major polypeptides form the backbone structure of epidermal filaments and the minor polypeptides play a role in its stabilization. 相似文献
19.
An HJ Lee HJ Jun SH Hwang SY Kim BC Kim K Lee KM Oh MK Kim J 《Bioprocess and biosystems engineering》2011,34(7):841-847
Lipase (LP) was immobilized on electrospun and ethanol-dispersed polystyrene–poly(styrene-co-maleic anhydride) (PS–PSMA) nanofibers
(EtOH-NF) in the form of enzyme precipitate coatings (EPCs). LP precipitate coatings (EPCs-LP) were prepared in a three-step
process, consisting of covalent attachment, LP precipitation, and crosslinking of precipitated LPs onto the covalently attached
LPs via glutaraldehyde treatment. The LP precipitation was performed by adding various concentrations of ammonium sulfate
(20–50%, w/v). EPCs-LP improved the LP activity and stability when compared to covalently attached LPs (CA-LP) and the enzyme
coatings of LPs (EC-LP) without the LP precipitation. For example, the use of 40% (w/v) ammonium sulfate resulted in EPC40-LP
with the highest activity, which was 4.0 and 3.6 times higher than those of CA-LP and EC-LP, respectively. After 165-day incubation
under rigorous shaking at 200 rpm, the residual activities of EPC50-LP were 0.5 μM/min mg of EtOH-NF, representing 113 and
75 times higher than those of CA-LP and EC-LP, respectively. When LP was partially purified via a simple ammonium sulfate
precipitation and dialysis, both activities and stabilities of EC-LP and EPC-LP could be marginally improved. It is anticipated
that the improved LP activity and stability in the form of EPCs would allow for their potential applications in various bioconversion
processes such as biodiesel production and ibuprofen resolution. 相似文献
20.
Phycocyanin is a major light harvesting accessory pigment of red algae and cyanobacteria. In the light of its many commercial applications in food and pharmaceutical industry, purity of the pigment plays a major role. Pharmaceutical industry demands a highly pure phycocyanin with A620/280 ratio of 4 and food industry a ratio of 2. In the present study phycocyanin was extracted in sodium phosphate buffer (pH 7) after macerating in liquid nitrogen. The crude phycocyanin thus extracted was precipitated with 50% ammonium sulphate, purified by dialysis and finally by gel filtration chromatography. Pure phycocyanin was finally obtained with an A620/A280 value of 4.98. 相似文献