共查询到20条相似文献,搜索用时 15 毫秒
1.
S Toyokawa T Takeda Y Kato K Wakabayashi Y Ogihara 《Chemical & pharmaceutical bulletin》1991,39(5):1244-1249
The complete amino acid sequence of a new abortifacient protein, karasurin, was determined. Karasurin, which was isolated from fresh root tubers of Trichosanthes kirilowii Maximowicz var, japonicum Kitamura (Cucurbitaceae), was a highly basic protein with pI 10.1 and molecular weight of 28,000. Intact karasurin was cleaved with cyanogen bromide, lysyl endopeptidase, formic acid and 2-(2'-nitrophenyl-sulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole), respectively. Cleavages with N-bromosuccinimide (NBS), trypsin and pepsin were performed for the fragments. The resultant peptide fragments were separated by gel filtration chromatography, reversed-phase high performance liquid chromatography (HPLC) or gel filtration HPLC following sequence analyses by automated Edman methods. Karasurin consists of 246 or 247 amino acid residues with a calculated molecular weight of 27,144 or 27,215 differing only at the C-terminus with the addition of alanyl residue. Two C-terminal sequences were identified as Asn-Asn-Met-OH and Asn-Asn-Met-Ala-OH by sequence analyses and hydrazinolysis, but there was no micro-heterogeneity in other peptides analysed. The sequence of karasurin revealed a considerable similarity to that of trichosanthin and alpha-trichosanthin, which are known as abortifacient, ribosome-inactivating and anti human immunodeficiency virus (HIV) (the virus causing acquired immunodeficiency syndrome (AIDS) proteins, with 93% and 98% identity, respectively. 相似文献
2.
S Toyokawa T Takeda Y Kato K Wakabayashi Y Ogihara 《Chemical & pharmaceutical bulletin》1991,39(8):2132-2134
Karasurin is an abortifacient plant protein isolated from fresh root tubers of Trichosanthes kirilowii Maximowicz var. japonicum Kitamrra (Cucurbitaceae). This study describes the presence of protein polymorphism in karasurin (karasurin-A and karasurin-B) separated by ion-exchange chromatography. Two components showed no differences in the molecular weight (ca. 28000), the amino acid composition, the neutral sugar content and part of the amino acid sequence. A unique difference was observed in their isoelectric points (10.1 for karasurin-A and 10.2 for karasurin-B). However, modifications of glycosylation or phosphorylation were not found. Biological assays for inducing mid-term abortion in pregnant mice and inhibition of the growth of BeWo cells gave no significant differences between them. The presence of protein polymorphism in karasurin, whose biological significance is not yet understood, is a first finding among several abortifacient proteins. 相似文献
3.
Hoffmann P Olayioye MA Moritz RL Lindeman GJ Visvader JE Simpson RJ Kemp BE 《Electrophoresis》2005,26(6):1029-1037
4.
Patrick P Berna Nathalie Berna Jerker Porath Sven Oscarsson 《Journal of chromatography. A》1998,800(2):26-159
Protein adsorption of human serum onto six different agarose-based chromatographic gels that were representative of the salt-promoted adsorbent family [octyl- and phenyl-Sepharose, mercaptoethanol–divinyl sulfone agarose (T gel), mercaptomethylene pyridine-derivatized agarose gel (MP gel), tricyanoaminopropene–divinyl sulfone agarose (DVS–TCP gel), tricyanoamino-propene–bisoxirane agarose (bisoxirane–TCP gel)] was studied in the presence of moderate or high concentrations of the water structuring salt, sodium sulfate. Study of the protein adsorption selectivity by two-dimensional gel electrophoresis revealed an opposed selectivity for hydrophobic interaction adsorbents and electron donor–acceptor adsorbents. The T gel, MP gel and TCP gels belonged to the electron donor–acceptor adsorbents, displaying a main selectivity for immunoglobulins, whereas octyl-Sepharose belonged to the hydrophobic adsorbents, displaying a main selectivity for ‘hydrophobic' proteins. Phenyl-Sepharose for its part was described as an example of a composite selectivity of both families. The conclusion of this work is two-fold: (1) hydrophobic interaction chromatography (HIC) and electron donor–acceptor chromatography (EDAC) have opposed protein selectivities and are both salt-promoted. As a main consequence, it means that high concentrations of a water-structuring salt can promote different types of weak molecular interactions, resulting in different protein adsorption selectivities: (2) thiophilic adsorption chromatography (TAC) should be renamed EDAC as similar protein selectivity is demonstrated for electron donor–acceptor ligand devoid of sulfur atoms. 相似文献
5.
Basic proteins in barley starchy endosperm from developing seeds were separated by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as partial extracts were analyzed. Edman degradation sequencing and immunological detection were performed after transfer of separated proteins onto membranes. Only one protein could be analyzed by N-terminal sequencing of blotted and separated proteins from the total extract. Fractionation of extracts was done using cation exchange chromatography, concanavalin A and heparin affinity chromatography. Internal sequences were determined after in-gel cleavage of proteins using trypsin or cyanogen bromide and separation of the fragments by reversed-phase chromatography or in a gel electrophoresis system for peptide separation. This resulted in a new protocol for obtaining internal sequences from proteins separated by 2-D electrophoresis. A total of 16 sequences, including nine internal sequences, were analyzed, permitting the identification of ten proteins, including five that appeared to have a blocked N-terminus. An additional protein was identified using immunological detection. Three protein sequences remained unidentified. Separated proteins were also analyzed with a glycan detection method. 相似文献
6.
Two lectins, an N-acetylgalactosamine-binding lectin, lectin-I, which reacts specifically with human erythrocytes of blood group A, and a galactose-binding lectin, lectin-II, which is specific for human blood group B erythrocytes, have been isolated and purified from rice bean, Phaseolus calcaratus syn. Vigna umbellata, by a salt solubility pH-dependent method, chromatofocusing and high-performance liquid chromatography. The homogeneity of the lectins was determined by liquid chromatography and polyacrylamide gel electrophoresis. The purified lectin-I of molecular mass 80,000 is possibly composed of two subunits of molecular mass ca. 18,000 and 22,000, respectively, whereas lectin-II of molecular mass 100,000 appears to be composed of a monomeric protein of molecular mass 25,000. One endogenous lectin-binding protein was also isolated and purified by liquid chromatography. The endogenous lectin-binding protein of molecular mass 40,000 affects the activity of the A-group specific lectin more than that of the B-group specific lectin. The endogenous lectin-binding protein appears to be composed of a monomeric protein of molecular mass 20,000. 相似文献
7.
To facilitate structural studies of receptor-interacting protein 3 (RIP3), we developed a large-scale expression system of
a glutathione-S-transferase (GST) fused with an 82 amino acid RIP3 protein inEscherichia coli. RIP3 truncation was subcloned into the pGEX-4T-1 vector and overexpressed in BL21(DE3)RIL cells. The soluble RIP3 protein
was successfully purified to homogeneity using GST tag, an anion-exchange column, and gel filtration chromatography. The purity,
identity, and conformation of the RIP3 protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis,
Western blotting, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, and fluorescence spectroscopic
studies. RIP3 showed dominance of the α-helix structure and temperature-dependent conformational change. 相似文献
8.
《液相色谱法及相关技术杂志》2012,35(11):2063-2071
Abstract Glutathione peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) was purified 3500-fold from rat liver with a yield of 42% using high performance liquid chromatography. The crucial purification step was size-exclusion chromatography on a Spherogel TSK-3000SW column, and the purified enzyme eluted as a single peak. The enzyme stained as a single band following SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be 105,000, and the subunit molecular weight determined by SDS-gel electrophoresis was 25,000. Polyacrylamide gel electrophoresis indicated five bands of protein with a broad of enzymatic activity. Isoelectric focusing resulted in a peak of enzymatic activity at pH 6.9 with a shoulder at pH 7.3. The specific activity of the purified enzyme was 1,100 μmol of NADPH oxidized per minute per milligram of protein. 相似文献
9.
High-yield two-step chromatographic procedure for purification of fatty acid-binding protein from human heart. 总被引:1,自引:0,他引:1
F A van Nieuwenhoven M M Vork D A Surtel A H Kleine G J van der Vusse J F Glatz 《Journal of chromatography. A》1991,570(1):173-179
A high-yield procedure for the purification of cytoplasmic fatty acid-binding protein from human heart (H-FABP) is described. H-FABP was purified by gel permeation chromatography on a Sephacryl S-200 column followed by anion-exchange chromatography on a Sepharose Q fast-flow column at pH 7.0. At this pH H-FABP binds strongly to the column and can be selectively eluted with a salt gradient. The two-step procedure showed a high degree of reproducibility. On average 50 mg of H-FABP was obtained from 150 g of human heart tissue, which corresponds to a recovery of about 50%. Purity was confirmed by gel electrophoresis and isoelectric focusing. Binding of oleic acid to purified H-FABP, using the Lipidex 1000 assay, revealed a maximal binding of 0.75 +/- 0.01 mol fatty acid/mol protein and a dissociation constant of 0.19 +/- 0.01 microM. 相似文献
10.
11.
The role of separation science in proteomics research. 总被引:22,自引:0,他引:22
H J Issaq 《Electrophoresis》2001,22(17):3629-3638
In the last few years there has been an increased effort into the separation, quantification and identification of all proteins in a cell or tissue. This is a review of the role gel electrophoresis, high performance liquid chromatography (HPLC), and capillary electrophoresis (CE) play in proteomics research. The capabilities and limitations of each separation technique have been pointed out. Instrumental strategies for the resolution of cell proteins which are based on efficient separation employing either a single high-resolution procedure or a multidimensional approach on-line or off-line, and a mass spectrometer for protein identification have been reviewed. A comparison of the advantages of multi-dimensional separations such as two-dimensional polyacrylamide gel electrophoresis, HPLC-HPLC, and HPLC-CE to the separation of cell and tissue proteins are discussed. Also, a discussion of novel approaches to protein concentration, separation, detection, and quantification is given. 相似文献
12.
Arachin and its molecular species (arachin I and arachin II) were separated and isolated. The number and kind of subunits of arachin, arachin I and arachin II were determined. Studies were carried out under different experimental conditions using slab gel electrophoresis, size-exclusion chromatography and reversed-phase high-performance liquid chromatography. Gel electrophoresis was done under varying concentrations of resolving gel. Tube gel as well as slab gel electrophoresis were used and continuous as well as discontinuous buffer systems were used for both types of electrophoresis. In addition, the subunits were separated by reversed-phase HPLC using a gradient program. Arachin and arachin II were found to have 12 subunits each while arachin I showed six subunits. The subunits of arachin I were allowed to reconstitute by removing SDS. Eight combinations were tried for studying the reconstitution pattern. Molecular weight and weight ratio in each case were also determined. 相似文献
13.
Cathepsin A (EC 3.4.16.1), a lysosomal carboxypeptidase, has been purified 1374-fold from pig kidney. Purification steps included concanavalin A-Sepharose and phenyl-Sepharose chromatography and chromatofocusing. The specific activity (16.9 U/mg) of the purified enzyme was significantly higher than previously reported values. The enzyme preparation appeared homogeneous when analyzed by non-denaturing polyacrylamide gel electrophoresis and was free of detectable protease contamination. The molecular mass (M(r) = 97,000), isoelectric point (5.0), and sensitivity to inhibitors were consistent with reported properties of cathepsin A. However, the previously reported three-peptide chain structure was not observed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol demonstrated that the enzyme is composed of two M(r) 47,000 subunits, each of which dissociate in the presence of 2-mercaptoethanol into two polypeptide chains of 19,000 and 31,000. 相似文献
14.
Matrix glycoproteins are among the main components that contribute to the properties of cartilage. In this article we report on the development of a rapid method for the fractionation and purification of a 92 kDa glycoprotein from chick sternal cartilage. The developed procedure involves ion-exchange chromatography on DEAE-Sephacel, gel permeation chromatography on Sepharose CL-6B and semi-preparative SDS-polyacrylamide gel electrophoresis. Identification of protein was performed by western blotting using specific antibodies and purity by capillary electrophoresis. The proposed method is superior to those previously published since it eliminates the step of density gradient centrifugation. 相似文献
15.
Prolactin and growth hormone are two peptide hormone with a very similar structure. A simple method is described for the simultaneous purification of these two peptides from canine pituitary extract by fast protein liquid chromatography. After extraction at pH 5.0 and 9.6 and anion-exchange chromatography on a MonoQ column, prolactin and growth hormone are then separated by gel filtration chromatography on two Superose columns coupled in series. The different fractions of the purification scheme are checked for the presence of the peptide hormones by sodium dodecyl sulphate gel electrophoresis in the Pharmacia PhastSystem. Each hormone is also characterized by its behaviour in a Western Blotting Detection System. 相似文献
16.
17.
Sachon E Mohammed S Bache N Jensen ON 《Rapid communications in mass spectrometry : RCM》2006,20(7):1127-1134
Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis. 相似文献
18.
Yassel Ramos Annia Gonzlez Patricia Sosa‐Acosta Yasset Perez‐Riverol Yairet García Lila Castellanos‐Serra Jeovanis Gil Aniel Snchez Luis J. Gonzlez Vladimir Besada 《Journal of separation science》2019,42(24):3712-3717
Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution. 相似文献
19.
Yu Xin Lanhua Liu Beizhan Chen Ling Zhang Yanjun Tong 《Journal of separation science》2015,38(9):1441-1448
An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel‐filtration, and thin‐layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax) of the affinity sorbent was 25 476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex “immobilized ligand‐aprotinin” (Kd) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel‐filtration chromatography revealed that the protein was a single polypeptide, and the purities were ~ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel‐filtration chromatography and thin‐layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15 907.1 ± 10.2 kallikrein inactivator unit/mg. 相似文献
20.
Microsequencing of proteins recorded in human two-dimensional gel protein databases. 总被引:1,自引:0,他引:1
H H Rasmussen J Van Damme M Puype B Gesser J E Celis J Vandekerckhove 《Electrophoresis》1991,12(11):873-882
Sixty-six human proteins recorded in the master transformed human epithelial amnion cells (AMA) (55) and keratinocyte (11) two-dimensional gel protein databases have been microsequenced since the last publication of the AMA database (Electrophoresis 1990, 12, 989-1071). Coomassie Brilliant Blue stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes and in situ digested with trypsin. The eluting peptides were separated by reversed-phase high performance liquid chromatography (HPLC), collected individually and sequenced. Computer searches using the FASTA and TFASTA programs from the Genetics Computer Group indicated that 29 of the analyzed polypeptides correspond to hitherto unknown proteins. 相似文献