共查询到20条相似文献,搜索用时 15 毫秒
1.
A P Zoli J F Beckers P Wouters-Ballman J Closset P Falmagne F Ectors 《Biology of reproduction》1991,45(1):1-10
A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942). 相似文献
2.
Purification and characterization of a novel proline-directed protein kinase from bovine brain. 总被引:12,自引:0,他引:12
A novel protein kinase which phosphorylates a synthetic peptide substrate (RRPDAHRTPNRAF) has been purified approximately 200,000-fold from bovine brain. This peptide contains the consensus sequence for phosphorylation by the p34cdc2 kinase. The purification procedure took advantage of the phenomenon that this novel brain kinase, in partially purified extracts, chromatographed on a gel filtration column as a high molecular weight complex which dissociated in buffer containing 1 M NaCl. The purified native enzyme was estimated to be approximately 63,000, and displayed two bands of M(r) = 33,000 and 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On Western immunoblot, the M(r) = 33,000 peptide reacted strongly with antibodies specific for a conserved amino-terminal sequence, weakly with antibodies to the conserved PSTAIRE sequence, and not at all with antibodies to the carboxyl terminus, of HeLa cell p34cdc2. The brain kinase and p34cdc2 were similar in displaying good activity toward the parent peptide substrate, but no activity toward peptide analogues in which the -T-P- motif was substituted with either -T-G- or -T-A-. Both kinases showed marked preference in phosphorylating a peptide derived from H1 histone (KTPKKAKKPKTPKKAKKL), and both kinases could be phosphorylated by the src-family tyrosine kinase, p56lyn, purified from bovine spleen. However, the brain kinase did not co-purify with a subunit having a molecular weight corresponding to known cyclins, nor did it undergo specific interaction with p13suc1 beads, suggesting that this enzyme is distinct from p34cdc2. 相似文献
3.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):442-447
AbstractA new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable. 相似文献
4.
Purification and characterization of a retinol-binding glycoprotein synthesized and secreted by bovine neural retina 总被引:5,自引:0,他引:5
S L Fong G I Liou R A Landers R A Alvarez C D Bridges 《The Journal of biological chemistry》1984,259(10):6534-6542
A retinol-binding glycoprotein ( IRBP ) was purified in milligram quantities from the extracellular matrix ( interphotoreceptor matrix) that occupies the subretinal space in bovine eyes. IRBP binds 2.2 molecules of all-trans retinol with a KD of approximately 10(-6) M. The holoprotein has lambda max at 280 nm (E1%1 cm = 10.99) and at 330 nm (E1%1 cm = 7.88). When freshly isolated from light-exposed eyes, IRBP contains up to 0.6 molecule of all-trans retinol, together with small amounts of the 11-cis and 13-cis isomers. IRBP also binds exogenous cholesterol, alpha-tocopherol, and all-trans retinoic acid, all of which are completely displaced by all trans retinol. The affinity of alpha-tocopherol for IRBP was at least several orders of magnitude less than that of all-trans retinol. IRBP contains 8.4% by weight of carbohydrate, which consists of sialic acid, neutral hexoses, and glucosamine in the molar ratio of approximately 1:3:2. No galactosamine was detected. Observations on the binding of 125I-labeled lectins to IRBP in sodium dodecyl sulfate-polyacrylamide gels before and after desialosylation suggest that at least one oligosaccharide chain is of the sialated biantennary complex type and contains fucose. The Mr of IRBP on calibrated size-exclusion columns averaged 249,000; on sodium dodecyl sulfate-polyacrylamide gels (with or without dithiothreitol) the apparent Mr was 144,000. IRBP exists in at least four isoelectric forms that bind concanavalin A and have pI values ranging from 4.4 to 4.8. Rabbit anti-bovine IRBP antiserum gave a single precipitin line against purified bovine IRBP , which showed a line of complete identity with crude bovine interphotoreceptor matrix and a line of partial identity with human interphotoreceptor matrix. The human material contains a prominent protein with lectin-binding properties similar to bovine IRBP but with a somewhat faster electrophoretic mobility. When isolated bovine neural retinas were incubated with 3H-labeled fucose, glucosamine, or leucine, a solitary labeled protein identified as IRBP was secreted into the medium. Labeled IRBP could not be detected in the medium when retinal pigment epithelium was incubated with these precursors under the same conditions. Neural retinas incubated with 3H-labeled leucine in the presence of tunicamycin secreted a form of IRBP that did not bind concanavalin A and had an Mr reduced by approximately 5,000. 相似文献
5.
Xiufen Yang Dewen Qiu Hongmei Zeng Jingjing Yuan Jianjun Mao 《World journal of microbiology & biotechnology》2009,25(11):2035-2042
Glycoprotein elicitor can induce plant resistance and become a potential agent for biological control of plant diseases. Here, a new glycoprotein elicitor was purified with the method of cold alcohol precipitation and anion exchange chromatography from the mycelium of Alternaria tenuissima strain JH505, which was identified on the basis of morphological features and sequence analysis of rDNA internal transcribed spacer. The protein showed a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) stained with silver and appeared one main protein peak in HPLC. The apparent molecular weight of the purified protein was 66 kDa and isoelectric point was about 4.27. This protein was identified as glycoprotein by glycoprotein staining Kit. Anthrone-colorimetric assay and Coomassie blue G-250 staining showed that carbohydrate and protein content was in a ratio of 1.75. After deglycosylation by trifluoromethane-sulfonic acid, this glycoprotein showed two bands on the SDS–PAGE, and which means the glycoprotein may have at least two glycosylation sites. The glycoprotein induced tobacco resistance against tobacco mosaic virus and enhanced wheat seedling growth at 15°C. The glycoprotein elicitor provided an effective way of alternative strategies for plant disease control. 相似文献
6.
《Process Biochemistry》2014,49(8):1362-1369
HEG-5, a novel glycoprotein with hemagglutinating activity, was firstly isolated and purified from the cultured mycelia of Hericium erinaceus CZ-2. SDS–PAGE, Native-PAGE and MALDI-TOF-MS proved that HEG-5 was a single band with the molecular weight of approximately 14.4 kDa. HEG-5 had the protein: polysaccharide ratio of approximately 10:1 (%/%) and contained d-glucose, l-rhamnose, d-galactose and d-mannose with a molar ratio of 1.00:1.09:2.45:7.14 in polysaccharide fraction. HEG-5 was an acidic glycoprotein with a PI value of 6.3 and the higher content of acidic amino acids (Asp, 12.42 ± 0.25% and Glu, 12.24 ± 0.26%) in protein fraction. FT-IR and NMR spectra revealed that HEG-5 contained the protein and carbohydrate portions with (1→4)-linked β-galactose residues and β-linked glucose residues. Circular dichroism (CD) demonstrated that HEG-5 was a β-sheet predominant glycoprotein. Hemagglutination assay proved it was a thermo-unstable glycoprotein. The HEG-5 structural novelty was finally presented by protein sequencing and modeling by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODEL Workspace service. 相似文献
7.
This paper reports the isolation and characterization from bovine milk of two proteins: angiogenin-1, a recently discovered angiogenin, and lactogenin, a novel protein. Both proteins were adsorbed on and eluted closely from CM-Sepharose and Mono S. Lactogenin possessed a molecular weight (17 kDa) slightly higher than that of angiogenin-1 (15 kDa). Lactogenin had a higher ribonucleolytic (RNase) activity than angiogenin-1 towards yeast transfer RNA (tRNA). The Km values estimated for the RNase activities of angiogenin-1 and lactogenin were 51 microM and 40 microM respectively. Both were specific for poly C. The optimal pH for the RNase activities of angiogenin-1 and lactogenin was 7.75 and 7.5 respectively. Comparison of the amino acid sequences of cyanogen bromide fragments and the pyroglutaminase-treated N-terminal fragment of lactogenin with the sequence of bovine liver RNase (RNase BL4) revealed identity in residues 3-22, 24, 26-27, 37, 41-44, 46-50, 54, 56, 63, 72-80, and 83. Considerable similarity to the N-terminal sequence of angiogenin-2 was also noted. Both lactogenin and angiogenin-1 inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) below 100 nM. 相似文献
8.
9.
Interferon-induced 2',5'-oligoadenylates are transiently produced during viral infection and are believed to play a role in the interferon-mediated inhibition of replication of at least some viruses. 2',5'-Oligoadenylates must be catabolized but are resistant to degradation by most known ribonucleases. A 2'-phosphodiesterase that degrades 2',5'-oligoadenylates was purified 1500-fold from a low speed homogenate of bovine spleen by precipitation at pH 5.2, ammonium sulfate fractionation, differential ultrafiltration, and successive chromatography on DEAE-Sephacel, hydroxylapatite, and a fast protein liquid chromatography Mono P column. No other 2-5A-degrading activity was observed during the purification procedure. The molecular mass of the enzyme estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 65,000. The enzyme is distinct from bovine spleen phosphodiesterase II. The 2'-phosphodiesterase cleaves 2',5'- and 3',5'-linked oligonucleotides, as well as branched oligoadenylate, A(2'pA)(3'pA), but appears to be most active on 3',5'-oligoribonucleotides. The enzyme cleaves 5'-AMP from the 2' terminus of 2',5'-oligoadenylates and appears to require a free 2' terminus and a 3'-oxygen on the penultimate nucleotide. Substrate length, 5'-phosphorylation, and base composition do not appear to be critical factors in determining enzyme activity. The effects of pH, Mg2+, Mn2+, EDTA, phosphate, 2-mercaptoethanol, and N-ethylmaleimide are also described. This enzyme may be involved in the catabolism of the interferon-induced 2',5'-oligoadenylates and other 2',5'-linked RNAs in the cell. 相似文献
10.
T F Zioncheck M L Harrison R L Geahlen 《The Journal of biological chemistry》1986,261(33):15637-15643
A protein-tyrosine kinase has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The purification procedure employs sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of Mr = 40,000. The p40 kinase contains an ATP-binding site as determined by photoaffinity labeling experiments and catalyzes an intramolecular autophosphorylation reaction that leads to its modification on tyrosine. Of several proteins tested only the cytoplasmic domain of the erythrocyte band 3 protein serves as a good substrate for p40 (Km = 12 microM). Increasing concentrations of NaCl stimulate the phosphorylation of angiotensin I, inhibit the phosphorylation of band 3, and have no effect on the autophosphorylation of p40. At low concentrations of NaCl, Mn2+ is the preferred divalent cation. Peptide mapping experiments indicate that p40 is distinct from pp60src and from the major phosphotyrosine containing proteins of T and B lymphocyte membranes. 相似文献
11.
Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas 总被引:7,自引:0,他引:7
The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma. 相似文献
12.
We have demonstrated previously that the Pz-peptide synthetic substrate is cleaved by two distinct spermatozoal peptidases, Pz-peptidases A and B. To facilitate further investigations, Pz-peptidase B was purified from bovine spermatozoa. The soluble extract from 81 grams of washed epididymal spermatozoa was fractionated by a five-step procedure consisting of anion-exchange, lectin affinity, hydrophobic interaction, chromatofocusing, and gel filtration chromatography. This method yielded 1 mg of 170-fold purified Pz-peptidase B with a 26% recovery. The purified Pz-peptidase B was electrophoretically homogeneous and possessed a monomeric molecular weight of 90,700. Isoelectric focusing revealed microheterogeneity with pIs ranging from 5.02 to 5.09. Pz-peptidase B was irreversibly inactivated at pH 3.5 or below, and activity was reduced at moderate ionic strengths. Hydrolysis of the Pz-peptide was maximal at pH 7.5. Pz-peptidase B was strongly inhibited by a metal chelator and phosphoramidon. Reactivation of metal-depleted enzyme by various metal ions suggested that Pz-peptidase B was a zinc metallopeptidase. Pz-peptidase B hydrolyzed a wide variety of synthetic substrates and physiologically activity peptides at the amino side of hydrophobic amino acids. These results established that Pz-peptidase B should be classified as a neutral metalloendopeptidase. Overall, the properties of Pz-peptidase B were very similar to those of previously described neutral metalloendopeptidases implicated in degradation of regulatory peptides. 相似文献
13.
The isolation from cancer patient serum of a glycoprotein (Cc) associated with the presence of a variety of malignancies was previously reported. Although preliminary chemical and physical data indicated that Cc was different from identified circulating glycoproteins, subsequent immunological studies suggested that it was closely related to alpha 1-acid glycoprotein. Further analysis revealed the presence of two components in some Cc preparations and prompted a re-examination of the isolation and characterization data. In the present study, Cc was purified by a modified protocol which involved the use of pleural fluid obtained from individuals with cancer, and an alpha 1-acid glycoprotein antibody column to remove contaminating alpha 1-acid glycoprotein. Typically, the material not retained by the antibody column gave a single band with Mr 53,000 when subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis. Amino terminal analysis revealed that the protein contained a blocked amino terminus, and carbohydrate analysis indicated that complex, asparagine-linked saccharide units were present. The product could be distinguished from alpha 1-acid glycoprotein and other previously described circulating glycoproteins by several criteria, including molecular weight, isoelectric point, and amino acid and carbohydrate composition. One of three preparations isolated had an apparent Mr of 59,000. Carbohydrate analysis as well as deglycosylation studies showed that the change in molecular weight was due to increased glycosylation. 相似文献
14.
Purification and characterization of osteopontin from human milk 总被引:3,自引:0,他引:3
Osteopontin (OPN) is expressed in many organs and tissues and has different biological properties related to different molecular forms in respect to size and posttranslational modifications. However, a purification procedure for authentic intact OPN as well as fragments of OPN from an accessible biological source is missing. A four-step procedure was used to purify OPN from human milk, based on its crystal growth inhibitory activity, including anion exchange chromatography, the elimination of casein, hydroxyapatite chromatography, and negative affinity chromatography. Purified OPN was further separated into its different molecular forms by means of a two-step procedure, involving size exclusion chromatography and reverse phase chromatography. A rabbit polyclonal antibody was raised to purified intact OPN and high M(r) OPN components; the immunoreactivity of both forms was almost equal when investigated by enzyme immunoassay (EIA). The procedures facilitate the purification of intact OPN and OPN fragments for purposes of standardization, preparation of monospecific antibodies, and functional studies. 相似文献
15.
Two major glycoproteins (PAS-6 and PAS-7) from bovine milk fat globule membrane were selectively extracted with urea and KCl, co-purified by repeated gel filtration on Sephacryl S-200 and then separated by affinity chromatography on concanavalin A-agarose column. The two purified glycoproteins showed a single band by SDS-PAGE, and their molecular masses were estimated to be 50 kDa for PAS-6 and 47 kDa for PAS-7. Both PAS-6 and PAS-7 were resolved several variants by analytical isoelectric focusing. These were shifted to a single band at pI 6.2 for PAS-6 and at pI 6.5 for PAS-7 by neuraminidase. PAS-6 contained 7.1% and PAS-7 5.5% of carbohydrate; the molar ratio of fucose:mannose:galactose:N-acetyl galactosamine:N-acetyl glucosamine:sialic acid was 1.0:3.0:2.0:6.1:5.0:1.3 for PAS-6 and 1.0:3.1:2.2:0:4.1:1.1 for PAS-7. Mild alkaline treatment and affinity to various lectins indicated that PAS-6 had O- and N-linked oligosaccharide chains, while PAS-7 had only the N-linked type. The major amino acid residues of PAS-6 were Glu, Ser and Gly, and those of PAS-7 were Asp, Glu, Gly and Leu. The N-terminal amino acids of both glycoproteins were blocked. PAS-6 and PAS-7 digested with trypsin had a different peptide map, two major peptides having the same retention time on HPLC and being common to PAS-6 and PAS-7 having the same amino acid sequences of H-Gln-Ser-Gly-Asn-Lys-Asn-Pro-Ser-Glu-Ile-Ser-OH and H-Ile-Phe-Pro-Gly-Asn-Met-Asp-Asn-Ser-His-Lys-OH. 相似文献
16.
Purification and MALDI-MS characterization of stressin, a stress-associated glycoprotein. 总被引:1,自引:0,他引:1
Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past years indicate they might also play an important role in the response to stress, a complex physiological response of the human organism to various threats. We have recently identified stressin, a human serum glycoprotein, which was found to be increased under stress conditions. Here we report the purification of stressin from sera of professional soldiers and partial characterization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Stressin was purified using a combination of ammonium sulfate precipitation, ion exchange chromatography, preparative gel electrophoresis and reverse-phase HPLC. It was found to be a highly glycosylated protein. Only 21.9 kDa (out of 36.7 kDa) was the protein part, whereas the remaining 40% of the mass originated from N-linked oligosaccharides. The carbohydrate part contained 12 sialic acids moieties, nearly 90% of which were lost due to post-source decay in the field-free tube. Tryptic fragments were produced from glycosylated and deglycosylated stressin, separated by reverse-phase HPLC and their exact molecular masses were determined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any already described protein. 相似文献
17.
Employing papain as the enzyme and agarose bound Ricinus communis agglutinin as the affinity gel, a glycoprotein has been isolated and purified from the surface of Ascaridia galli. The glycoprotein shows an apparent molecular weight of 68 kilo daltons and contains fucose, galactose, rhamnose and glucosamine as sugar moieties. Only 2% of its entire molecule has been found to possess alpha-helical configuration. 相似文献
18.
John E. Hesketh Noelle Virmaux Paul Mandel 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,542(1):39-46
Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin. 相似文献
19.
20.
Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme. 相似文献