共查询到20条相似文献,搜索用时 15 毫秒
1.
Fibroblast growth factors are proteins which play a major role, in vitro and in vivo, in the control of cellular growth and differentiation of a large number of cells. Biological activities of these factors are mediated by the interaction with specific membrane receptors. Previous studies indicated that the apparent molecular weight of a family of these receptors for the basic form of Fibroblast Growth Factor (bFGF), ranges from 125 to 165 kDa according to cell species and types. We have purified this family of receptors from bovine brain. We first set up a radioreceptor assay to detect receptors throughout the purification by measuring its ability to inhibit the fixation of radiolabeled bFGF to insolubilized membranes from bovine brain. The purification was also monitored by using cross-linking reagents in order to allow the visualization of radiolabeled bFGF bound to its receptor. The first purification steps involved 2 anion-exchange chromatographic steps, DEAE Trysacryl and FPLC Mono Q, and yielded an enrichment over 500 fold. Affinity chromatography with bFGF immobilized on Sepharose 4B was then performed. Covalent fixation of bFGF to the Sepharose matrix was carried out in presence of N-acetylated heparin in order to protect the recognition site for bFGF on its receptor. These 3 chromatographic steps yielded only 2 bands of apparent molecular weight of 100 kDa and 135 kDa as detected by electrophoresis. These 2 bands are also detected after chromatography on immobilized wheat germ agglutinin hence confirming the presence of carbohydrates on bFGF receptors. 相似文献
2.
A rapid and highly efficient procedure for purification of kininase II from human seminal plasma is described. After ultra centrifugation, the enzyme was purified by gel filtration on Sepharose 6B CL and ion exchange chromatography followed by affinity chromatography of EDTA-inhibited enzyme on bradykinin-Sepharose. The enzyme was specifically inhibited by Captopril and BPP9a but not by phosphoramidon. PAGE in the presence of sodium dodecyl sulfate under reducing conditions resulted in two major protein bands with apparent molecular masses of about 55 kDa and 65 kDa and two faint protein bands at higher molecular masses. Antibodies raised against the major protein bands showed full cross reactivity with all four protein bands. The presented data indicate that kininase II consists of subunits. 相似文献
3.
Purification of an active opioid-binding protein from bovine striatum 总被引:12,自引:0,他引:12
T L Gioannini A D Howard J M Hiller E J Simon 《The Journal of biological chemistry》1985,260(28):15117-15121
We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors. 相似文献
4.
K Shailubhai B Dong-Yu E S Saxena I K Vijay 《The Journal of biological chemistry》1988,263(31):15964-15972
The GlcNAc-1-P-transferase that initiates the dolichol cycle for the biosynthesis of asparagine-linked glycoproteins has been purified from the lactating bovine mammary gland. After solubilization from microsomes with 0.25% Nonidet P-40, the enzyme activity was stabilized with 20% glycerol, 20 micrograms/ml phosphatidylglycerol, 5 microM dolichol phosphate, and 2.5 microM UDP-GlcNAc. The purification protocol involved (NH4)2SO4 precipitation, gel filtration on Sephacryl S-300, DEAE-TSK, and hydroxylapatite chromatography. The purified enzyme was devoid of several readily detectable glycosyltransferases of the dolichol cycle. It showed two bands (A, 50 kDa and B, 46 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after either Coomassie Blue or silver staining. Antisera (anti-A and anti-B) raised against individual bands A and B inhibited the enzyme activity in solubilized microsomes. Each of the partially purified antibodies recognizes both bands A and B on Western blots of the enzyme; with the solubilized microsomes, the antibodies also recognize an additional polypeptide of approximately 70 kDa. When radioiodinated microsomes were immunoprecipitated with anti-B and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, again bands of 46, 50, and 70 kDa were observed. The peptide mapping of 50 and 46 kDa bands of the purified enzyme by chemical cleavage with N-chlorosuccinimide gave similar fragmentation patterns. The results indicate that either 70 kDa band is a precursor form of the enzyme or this polypeptide, representing the native enzyme or its subunit, is proteolyzed to smaller, enzymatically active peptide(s) of 50 and 46 kDa during purification despite the inclusion of several inhibitors against serine-proteases in all buffers used for tissue homogenization and enzyme purification. A number of properties of the purified enzyme, including its specific activation by Man-P-Dol were also characterized. 相似文献
5.
Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line 总被引:1,自引:0,他引:1
Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT. 相似文献
6.
Identification of Toxoplasma gondii proteins binding human lactoferrin: a new aspect of rhoptry proteins function 总被引:1,自引:0,他引:1
In this paper, we report on the isolation, purification and identification of two Toxoplasma gondii membrane proteins binding human lactoferrin. Parasite membrane proteins were isolated using the commercial Mem-PER Eukaryotic Membrane Protein Extraction System. After purification by lactoferrin affinity chromatography, three protein bands were detected with the molecular mass of 74, 63 and 58 kDa, two of which (63 and 58 kDa) specifically bound biotin labeled human lactoferrin as examined by competitive inhibition. Further identification of latter proteins by ESI/MS/MS amino acid sequencing technique revealed those proteins as Toxoplasma ROP4 (band 63 kDa) and ROP2 (band 58 kDa) antigens known to be involved in many mechanisms essential for the parasite pathogenicity, including host lactoferrin acquisition as determined in this study. 相似文献
7.
Partial purification of rat liver microsomal glucose-6-phosphatase on hydroxylapatite 总被引:1,自引:0,他引:1
Glucose-6-phosphatase was effectively solubilized from rat liver-microsomal membrane by the nonionic detergent Renex 690 in the presence of 0.6M sodium chloride. Subsequent separation on hydroxylapatite proved to be a successful and rapid initial step towards the purification of this enzyme. Glucose-6-phosphatase appeared in the colourless void volume with a yield of about 40-50%. The specific activity in the pooled void volume was 3-4 U/mg protein representing an enrichment of 30- to 40-fold. The best final specific activity obtained in an enriched fraction was 6.7 U/mg protein. Analysis of the pooled glucose-6-phosphatase-enriched fraction by SDS electrophoresis revealed 2 dominant protein bands with the apparent molecular mass of 17 and 18.5 kDa and few weak protein bands in the range of 21 to 42 kDa. 相似文献
8.
The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%. 相似文献
9.
Horn JM Lehman JA Alter G Horwitz J Gomez-Cambronero J 《Biochimica et biophysica acta》2001,1530(1):97-110
Utilizing the transphosphatidylation reaction catalyzed by phospholipase D (PLD) in the presence of a primary alcohol and the short-chain phospholipid PC8, we have characterized the enzyme from human neutrophils. A pH optimum of 7.8-8.0 was determined. PIP(2), EDTA/EGTA, and ATP were found to enhance basal PLD activity in vitro. Inhibitory elements were: oleate, Triton X-100, n-octyl-beta-glucopyranoside, divalent cations, GTPgammaS and H(2)O(2). The apparent K(m) for the butanol substrate was 0.1 mM and the V(max) was 6.0 nmol mg(-1) h(-1). Immunochemical analysis by anti-pan PLD antibodies revealed a neutrophil PLD of approximately 90 kDa and other bands recognized minimally by anti-PLD1 or anti-PLD2 antibodies. The 90-kDa protein is tyrosine-phosphorylated upon cell stimulation with GM-CSF and formyl-Met-Leu-Phe. Protein partial purification using column liquid chromatography was performed after cell subfractionation. Based on the enzyme's regulatory and inhibitory factors, and its molecular weight, these data indicate an enzyme isoform that might be different from the mammalian PLD1/2 forms described earlier. The present results lay the foundation for further purification of this granulocyte PLD isoform. 相似文献
10.
Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis 总被引:1,自引:0,他引:1
A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry. 相似文献
11.
The fetal isoenzyme of thymidine kinase was purified to apparent homogeneity from cytosols of rat fetuses liver. A two-step purification including anion exchange chromatography and affinity chromatography was developed. The purified enzyme appears as oligomeric with a relative molecular weight of 71 kDa. In denaturing media its molecular weight was 24 kDa, and its pHi 8.3. 相似文献
12.
Guy Baron Anne Madec-baron Suzanne Sampérez Pierre Jouan 《Preparative biochemistry & biotechnology》2013,43(3-4):241-256
The fetal isoenzyme of thymidine kinase was purified to apparent homogeneity from cytosols of rat fetuses liver. A two-step purification including anion exchange chromatography and affinity chromatography was developed. The purified enzyme appears as oligomeric with a relative molecular weight of 71 kDa. In denaturing media its molecular weight was 24 kDa, and its pHi 8.3. 相似文献
13.
Yutaka Hirashima Akhlaq A. Farooqui John S. Mills Lloyd A. Horrocks 《Journal of neurochemistry》1992,59(2):708-714
Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Ma.Carmen Santiago-Santos Teresa Ponce-Noyola Roxana Olvera-Ramrez Jaime Ortega-Lpez Rosa Olivia Caizares-Villanueva 《Process Biochemistry》2004,39(12):2047-2052
The extraction and purification of phycocyanin from Calothrix sp., cyanobacteria isolated from rice fields in Cuernavaca, Morelos, Mexico is described. Phycocyanin was extracted with 2 mg of lysozyme/g wet biomass, and purified by anion chromatography using Q-Sepharose fast-flow (Pharmacia®, 1.5 cm×10 cm) column and hydrophobic interaction chromatography with methyl macro-prep (Bio-Rad®, 1.5 cm×20 cm) column. The purified protein showed a pI of 5.2 and has two subunits with apparent molecular mass of 21–17 kDa each. The estimated molecular mass of native purified phycocyanin was 114 kDa, suggesting a stereochemistry of (β)3. 相似文献
15.
This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the "soluble" enzyme subtypes and their "particulate" counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle. 相似文献
16.
AMP-deaminase was purified from skeletal muscle of rat by the affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) has shown three protein bands on each step of purification. One of them corresponds to the subunit of tetrameric AMP-deaminase molecule with molecular weight of 76 kDa and two others--to the protein subunit with molecular weight of 42 and 33 kDa. Repeated SDS-PAGE of the main subunit band has revealed again all these protein bands. The data obtained indicate that AMP-deaminase subunit of 76 kDa is able to dissociate on two polypeptide chains with similar values of molecular weights in the presence of SDS. 相似文献
17.
Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms. 相似文献
18.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1994,1204(2):243-249
Selenoprotein P was partially purified (> 1000-fold) from human plasma in four chromatographic steps using 75Se-labeled selenoprotein P secreted by HepG2 cells in culture as a marker. The purified preparation was injected into mice and monoclonal antibodies, which precipitated the labeled protein, were generated. Neither of two different monoclonal antibodies had cross-reactivity with plasma from five animal species. Antibodies were coupled to agarose, and selenoprotein P was purified from human plasma by immunoaffinity chromatography followed by chromatography on heparin agarose. With two different matrix-bound monoclonal antibodies, the purification procedure gave two bands on SDS-PAGE with mobilities corresponding to 61 and 55 kDa. Both bands stained for carbohydrate and showed increased electrophoretic mobility after enzymatic deglycosylation. Immunoaffinity chromatography removed approx. one-third of the selenium from plasma or 0.4 μmol Se/l at a total selenium concentration of 1.1 μmol/l, indicating that selenoprotein P constituted this proportion of total plasma selenium in healthy US blood donors. 相似文献
19.
A membrane-bound phosphatidylinositol 4-kinase (PtdIns kinase) has been purified to apparent homogeneity from human erythrocytes. Enzyme activity was solubilized from urea-KCl-stripped, inside-out membrane vesicles by 3% Triton X-100. Purification to apparent homogeneity was accomplished by cation-exchange chromatography on phosphocellulose, followed by heparin-acrylamide chromatography. This resulted in a nearly 3900-fold purification of PtdIns kinase activity to a specific activity of 44 nmol min-1 mg-1. The purified enzyme has an Mr of 59,000 on silver-stained SDS-PAGE; however, many preparations also contain 54 kDa and 50 kDa proteins which are related to the 59 kDa protein and have PtdIns kinase activity. Kinetic analysis of the PtdIns kinase indicate apparent Km values of 40 and 35 microM for phosphatidylinositol and ATP, respectively. The purified enzyme has been reconstituted into phospholipid liposomes and shown to phosphorylate phosphatidylinositol. 相似文献
20.
Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain. 总被引:1,自引:0,他引:1
The sodium- and potassium-coupled L-glutamate transporter from rat brain has been purified to near homogeneity by reconstitution of transport as an assay, assuming that inactivated and active transporters cochromatograph. The purification steps involve lectin chromatography of the membrane proteins solubilized with 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fractionation on hydroxylapatite, and ion-exchange chromatography. The specific activity is increased 30-fold. The actual purification is higher since 3-5-fold inactivation occurs during the purification. The efficiency of reconstitution was about 20%. The properties of the pure transporter are fully preserved. They include ion dependence, electrogenicity, affinity, substrate specificity, and stereospecificity. Sodium dodecyl sulfate-polyacrylamide electrophoresis revealed one main band with an apparent molecular mass of around 80 kDa and a few minor bands. Comparison of polypeptide composition with L-glutamate transport activity throughout the fractionation procedure reveals that only the 80-kDa band can be correlated with activity. The GABA transporter, which has the same apparent molecular mass (Radian et al., 1986), is separated from it during the last two purification steps. Immunoblot experiments reveal that the antibodies against the GABA transporter only reacted with fractions exhibiting GABA transport activity and not with those containing the glutamate transporter. We conclude that the 80-kDa band represents the functional sodium- and potassium-coupled L-glutamate transporter. 相似文献