共查询到20条相似文献,搜索用时 31 毫秒
1.
Yamashiro K Takasu N Inui T Kouki T Ochi Y Kajita Y Sato Y Nagata A 《Life sciences》1999,64(13):1109-1116
In the previous reports, we have demonstrated (1) that polyethylene glycol (PEG)(5%) augmented TSAb (thyroid stimulating antibody)-stimulated cAMP responses of porcine thyroid cells, and (2) that fragments from papain-digested TSBAb (thyroid stimulation blocking antibody) could stimulate thyroid cAMP synthesis. Thus, we studied the effect of 5% PEG on cAMP responses stimulated by the protease-digested TSAb- or TSBAb-fragments. Stimulatory effect of 5% PEG on cAMP production by Fab fragment (Mr 50 KDa) and the retarded fraction (Mr 20 KDa) from the gel-filtration on Sephadex G-100 using papain-digested TSAb-IgG unbound to Protein A-Sepharose was observed. Similar stimulatory effect of 5% PEG on the second fraction (Fc with trace amounts of Fab) in the gel-filtration on Sephadex G-100 using papain digested TSAb-IgG bound to Protein A-Sepharose was observed. Stimulatory effect of PEG on the second fraction was derived from Fab fragment. PEG (5%) also showed stimulatory effect on cAMP production by F(ab')2 fragment (Mr 100 KDa) from the gel-filtration on Sephadex G-100 using pepsin-digested TSAb-IgG unbound to Protein A-Sepharose. PEG (5%) augmented cAMP responses by both Fab and the retarded fractions from the gel-filtration using papain-digested TSBAb-IgG unbound to Protein A when these fractions could stimulate cAMP synthesis. In conclusion, PEG (5%) augments cAMP responses stimulated by F(ab')2, Fab and the smaller molecular components (Mr 20 KDa) separated from protease-digested TSAb-IgG. PEG also augments cAMP responses stimulated by Fab and the smaller molecular components with thyroid stimulating activity separated from papain-digested TSBAb-IgG. 相似文献
2.
A protein which facilitates assembly of nucleosome-like structures in vitro in mammalian cells 总被引:3,自引:0,他引:3
An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells. The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation. In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons. When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented. The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts. Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction. The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease. 相似文献
3.
Extensive charging of transfer ribonucleic acid by bean leaf extracts in vitro 总被引:1,自引:1,他引:0
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1. Phenol was effectively removed from aqueous extracts of RNA by chromatography on Sephadex G-50. 2. Elution of tRNA from Sephadex G-50 columns at pH7.6 was shown to remove 91% of the endogenously bound amino acids. 3. tRNA prepared without recourse to ethanolic precipitation was capable of accepting much greater amounts of amino acids than could redissolved samples of precipitated tRNA. 4. Aminoacyl-tRNA synthetase enzymes were partially purified with calcium phosphate gel. Elution of enzymes from the gel at pH6.5 yielded a fraction having phenylalanine- and alanine-charging activity, but no aspartate-, lysine- or proline-charging activity, whereas elution at pH7.6 gave a fraction having aspartate-, lysine- and proline-charging activity but no phenylalanine- or alanine-charging activity. 5. By using partially synthetase enzymes and tRNA eluted from DEAE-Sephadex A-50 columns, 52% of the theoretical maximum of aminoacyl-tRNA synthesis was obtained in vitro. 相似文献
4.
Jelínková P Manásková P Tichá M Jonáková V 《International journal of biological macromolecules》2003,32(3-5):99-107
Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (Mr>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with Mr 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown. 相似文献
5.
M Privat de Garilhe M Vedel C Tempête C Federici E Garcia-Giralt 《Biomedicine / [publiée pour l'A.A.I.C.I.G.]》1980,32(3):148-154
Lymphoid chalone extracts, obtained from bovine spleen were purified on chromatographic columns: in the first step of the procedure, exclusion chromatography on Sephadex G75, and ion exchange chromatography on DEAE Sephadex were applied. A purified, active material was thus obtained. Upon thin layer chromatography, this fraction was shown to contain 3-4 UV absorbing components and 2 ninhydrin positive components. Further purification on Biogel P2, on Sephadex G10 and preparative thin layer chromatography, showed that the biological activity was located in small molecular weight components which belong to the polypeptide series. The ultraviolet absorbing components were identified as known nucleosides. The purified material shows an inhibitory effect on the uptake of 3H-thymidine by lymphocytes, as well as on mitogen induced lymphocyte transformation and haemolysin plaque forming cells. Furthermore, this inhibitory effect is specific for lymphoid cells. 相似文献
6.
Properties of cytosolic components activating rat hepatic 5'' [corrected]-deiodination in the presence of NADPH. 总被引:5,自引:5,他引:0
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The effects of cytosol, NADPH and reduced glutathione (GSH) on the activity of 5'-deiodinase were studied by using washed hepatic microsomes from normal fed rats. Cytosol alone had little stimulatory effect on the activation of microsomal 5'-deiodinase. NADPH had no stimulatory effect on the microsomal 5'-deiodinase unless cytosol was added. 5'-deiodinase activity was greatly enhanced by the simultaneous addition of NADPH and cytosol (P less than 0.001); this was significantly higher than that with either NADPH or cytosol alone (P less than 0.001). GSH was active in stimulating the enzyme activity in the absence of cytosol, but the activity of 5'-deiodinase with 62 microM-NADPH in the presence of cytosol was significantly higher than that with 250 microM-GSH in the presence of the same concentration of cytosol (P less than 0.001). The properties of the cytosolic components essential for the NADPH-dependent activation of microsomal 5'-deiodinase independent of a glutathione/glutathione reductase system were further assessed using Sephadex G-50 column chromatography to yield three cytosolic fractions (A, B and C), wherein A represents pooled fractions near the void volume, B pooled fractions of intermediate Mr (approx. 13 000), and C of low Mr (approx. 300) containing glutathione. In the presence of NADPH (1 mM), the 5'-deiodination rate by hepatic washed microsomes is greatly increased if both A and B are added and is a function of the concentrations of A, B, washed microsomes and NADPH. A is heat-labile, whereas B is heat-stable and non-dialysable. These observations provide the first evidence of an NADPH-dependent cytosolic reductase system not involving glutathione which stimulates microsomal 5'-deiodinase of normal rat liver. The present data are consistent with a deiodination mechanism involving mediation by a reductase (other than glutathione reductase) in fraction A of an NADPH-dependent reduction of a hydrogen acceptor in fraction B, followed by reduction of oxidized microsomal deiodinase by the reduced acceptor (component in fraction B). 相似文献
7.
1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts. 相似文献
8.
I S Thakur 《Biochemistry international》1991,23(3):449-459
Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen. 相似文献
9.
N-Acetyl-D-glucosamine-binding lectin was isolated and purified from rice by ammonium sulphate fractionation and affinity
chromatography using N-acetyl-D-glucosamine linked Sepharose 6B column. It gave a single hand on Polyacrylamide disc gel.
It was identified as a glycoprotein. The purified lectin dissociated into two components on Sephadex G-100 column chromatography,-a
higher molecular weight fraction not containing any carbohydrate and a lower molecular weight glycoprotein fraction. The apparent
molecular weights of these fractions were 85,000 and 14,500. The lectin agglutinated erythrocytes of human A,B,O groups and
of several other mammals and its activity was inhibited only by N-acetyl-D-glucosamine. The glycopeptide isolated by pronase
digestion of the lectin was homogeneous and did not possess agglutinating activity. It contained about 10% carbohydrate of
which xylose, arabinose and glucose were the major components. 相似文献
10.
Abstract— The venom of homet ( Vespa insularis) was separated into three components by gel filtration on Sephadex G-50, Sephadex G-25 and Sephadex G-10. The effect of each component on crustacean neuromuscular junctions was studied electrophysiologically using intracellular recordings. Two components (Fractions D and E) suppressed the postsynaptic potentials; Fraction D preferentially depressed excitatory postsynaptic potentials (epsps) without affecting inhibitory postsynaptic potentials (ipsps), and fraction E depressed both epsps and ipsps with concurrent decrease in membrane resistance. Another component (fraction F) augmented both epsps and ipsps. Fractions D and F were possibly acting presynaptically while fraction E was acting on the postsynaptic membrane. The active substances in fraction D, E and F were separated on TLC and the active substance in fraction F was confirmed to be serotonin. The active substances in fractions D and E are assumed to be peptides from the results of peptidase digestion. The effect of fraction D was degraded by treatment with trypsin but not by chymotrypsin nor carboxypeptidase A and B. On the other hand, the effect of fraction E was degraded by chymotrypsin and carboxypeptidase B but not by trypsin nor carboxypeptidase A. 相似文献
11.
Eosinophil and neutrophil chemotactic activities of adult worm extracts of Schistosoma japonicum in vivo and in vitro 总被引:1,自引:0,他引:1
Large numbers of eosinophils and neutrophils attracted to the soluble extract of Schistosoma japonicum adult worms (SjAW-ext) were detected at the injection site of normal guinea pig skin. Eosinophil and neutrophil chemotactic activities were also confirmed in in vitro assay by using blind-well chambers with Millipore filters in dose-dependent fashion. Two components of SjAW-ext showed eosinophil chemotactic activity; one was in the high molecular weight fraction (JAE-H), estimated to be more than 440,000 daltons, the other in the low molecular weight fraction (JAE-L) obtained by Sephadex G-200 gel filtration. High neutrophil chemotactic activity was detected in the JAE-L. These eosinophil and neutrophil chemotactic activities were also detected in culture fluid of S. japonicum adult worms. Eosinophil chemotactic factor (ECF) of JAE-H was stable to heating (100 C, 30 min) and pronase digestion, but completely destroyed by periodate oxidation. It is suggested that the ECF of JAE-H is a glycoprotein. JAE-L was also stable to heating (56 and 100 C, 30 min) and pronase digestion for eosinophil chemotaxis. Possible roles of those activities in schistosome infections are discussed. 相似文献
12.
V S Samsonova N V Kholchev E A Mamaeva L S Kuznetsova G V Shna?der 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1975,(6):73-78
Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties. 相似文献
13.
Proteins were extracted from rat liver ribosomal subunits with ethanol and ammonium chloride. The extract from the 40S subunit contained mainly S25, but smaller amounts of a number of other proteins were found as well; the extract from the 60S subparticle had L16 in addition to P1, P2, S25, and several other proteins. S25 and L16 had not been purified before. The former was isolated from the ethanol-ammonium chloride extract by stepwise elution from carboxymethylcellulose with LiCl, chromatography on phosphocellulose, and filtration through Sephadex G-75; L16 was purified by elution from carboxymethylcellulose with LiCl (in steps). The molecular weight of the two proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; and amino acid composition was determined also. 相似文献
14.
Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins 总被引:5,自引:3,他引:2
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Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis. 相似文献
15.
Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid 总被引:5,自引:3,他引:2
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1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature. 相似文献
16.
Ethylene inducing proteins were partially purified and characterized from the cell wall digesting enzyme mixture, Cellulysin. Purification included binding to Sephacryl S-200, isoelectric focusing, molecular sieving on Sephadex G-75, agarose electrophoresis, and sizing using a Superose 12 column. At least three active proteins were obtained from the Sephadex G-75 fraction that move towards the cathode during nondenaturing agarose electrophoresis. These three protein fractions separated by preparative agarose electrophoresis contain polypeptide patterns that are very similar on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions contain three main Coomassie blue stained bands of about 10, 14, and 18 kilodaltons. Gel filtration of the major fraction on a Superose 12 column yields an active peak with an apparent molecular weight of 27,000. Proteolytic enzymes, in the presence of urea, destroy the ethylene inducing activity. We conclude that the ethylene inducing factor (EIF) that we have isolated from Cellulysin is protein. Similar ethylene inducing factors are present in Cellulase RS. Ethylene inducing components from pectinase, Pectolyase, and Rhozyme do not bind to Sephacryl like EIF from Cellulysin. Thus, the components responsible for the ethylene inducing activity in these latter enzyme preparations differ from that of EIF. 相似文献
17.
A R Sheth L R Joshi S B Moodbidri S S Rao 《Journal of reproduction and fertility. Supplement》1979,(26):71-85
This communication presents evidence for the existence in the ovine testis of proteinaceous factors which suppress LH as well as FSH. Isolation of these factors has been achieved by using three different procedures: cytosol preparation, metaphosphoric acid extraction and ultrafiltration. Chromatography of cytosol or metaphosphoric acid extract on Sephadex G-75 resulted in separation into three protein fractions designated as G-75-I, II and III in order of their elution. When administered to castrated male rats, Fraction G-75-I suppressed circulatory levels of LH (53% inhibition, P less than 0.05) without altering FSH. The most retarded fraction, G-75-III, suppressed FSH (29% inhibition, P less than 0.001) without any concomitant change in LH. When fraction G-75-III was further fractionated on Sephadex G-25, three components were found and two, G-25-II and G-25-III, were biologically active. These fractions were homogeneous on polyacrylamide disc-gel electrophoresis. The FSH-suppressing factor (inhibin) was heat labile and susceptible to trypsin digestion, indicating that it is proteinaceous. Treatment with urea did not reveal any subunits. The molecular weight of this factor, as determined by gel filtration and SDS-urea gel electrophoresis was estimated to be around 1400-1500. The absence of sialic acid and the molecular weight data suggested that the isolated material was a simple protein and probably a small peptide. Gel filtration on Sephadex G-75 of the metaphosphoric acid extracts of liver, kidney, testis and ovary revealed an identical elution pattern for ovarian and testicular inhibin. 相似文献
18.
Separation of the Proteolytic and Streptolytic Components of a Streptomyces albus Enzyme Preparation and Their Effect on Streptococcal Cells 总被引:1,自引:0,他引:1
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The complex of enzymes elaborated by a strain of Streptomyces albus was separated by Sephadex G-75 column chromatography. Two major and one minor proteolytic peaks and one streptolytic peak were obtained. The streptolytic fraction alone did not cause lysis of the cells, but was highly effective in the presence of small amounts of trypsin or of the proteolytic components of the enzyme complex. Treatment of cells with the purified streptolytic fraction effected release of group carbohydrate and of a trypsin and pepsin-resistant protein antigen which reacted with type-specific antisera. 相似文献
19.
M Sugiyama M Sakamoto H Mochizuki O Nimi R Nomi 《Journal of general microbiology》1983,129(6):1683-1687
Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5'-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mM and 0.4 mM, respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin. 相似文献
20.
Ikunori Koshiyama 《Bioscience, biotechnology, and biochemistry》2013,77(7):646-650
Carbohydrate components in the 7S protein from soybean casein fraction were found to be mannose and hexosamine. The former was identified by paper and starch-column chromatographies and its content was approximately 4% per protein. The latter, hexosamine, was contained about 1.2% per protein.Mannose was considered as an integral constituent of the 7S protein from the data of heat and acid denaturation, paper electrophoresis and column chromatography with Sephadex G-200. 相似文献