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1.
A 200-kDa peanut agglutinin (PNA)-binding glycoprotein, gp200, has been purified and partially characterized from the human embryonal carcinoma cell line, HT-E (833k). Tissue distribution analysis of this molecule by lectin blotting with PNA of detergent-extracted proteins from human cell lines and tissues demonstrated expression limited to nonseminomatous germ cell tumors. The 200-kDa protein was purified with lectin affinity and gel filtration chromatography. Purification to apparent homogeneity was demonstrated by one- and two-dimensional gel electrophoresis. Characterization of gp200 revealed it to be a surface integral membrane glycoprotein; however, gp200 could also be purified from the culture media of EC cells, suggesting gp200 has an extracellular role. The carbohydrate groups of gp200 are N-linked and partially sialylated and contain terminal galactose residues. These initial studies suggest that the PNA-defined glycoprotein, gp200, is a candidate for a nonseminomatous germ cell tumor marker. 相似文献
2.
Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD. 相似文献
3.
Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. 相似文献
4.
A glycogen synthase, designated GS II, which occurs in a protein/carbohydrate complex has been purified from Ascaris suum muscle. The purified GS-II complex which is eluted from concanavalin-A--Sepharose contains proteins with Mr 140,000 and 66,000 and a glycoprotein with a carbohydrate/protein mass ratio of 3:1. GS II activity was totally dependent on glucose 6-phosphate, but exogenous glycogen was not required for polysaccharide synthesis. The GS-II complex was not phosphorylated by cyclic-AMP-dependent protein kinase, and antibodies to the protein and carbohydrate components of GS II did not cross react with the purified cyclic-AMP-regulated glycogen synthase (GS I) from A. suum muscle. Polysaccharide which was synthesized de novo by the complex was added to the large-molecular-mass glycoprotein in GS II. The glycogen-like character of the newly synthesized polysaccharide was confirmed by the observation that glycogen phosphorylase utilized the polymer as substrate in both the synthesis and degradation reactions. A model is discussed in which a core glycoprotein serves as the substrate for a glycogen synthase which is distinctly different from GS I. 相似文献
5.
Isolation and characterization of a collagen-binding glycoprotein from chondrocyte membranes 总被引:18,自引:2,他引:16
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A collagen-binding glycoprotein was isolated from purified chick chondrocyte surface membranes by affinity chromatography on type II collagen-Sepharose. The purified glycoprotein has an apparent mol. wt. of 31,000 and binds to native chick collagen types I, II, III, V and M. Although it contains 30% carbohydrates, the majority of which is fucose, it is hydrophobic and soluble only in detergents. The integral membrane protein character of the 31-K protein became apparent from its ability to insert into lecithin vesicles. Liposome-inserted 31-K protein binds 125I-labelled type II collagen in the presence of 0.5 M NaCl, while detergent-solubilized 31-K protein is dissociated from type II collagen by 0.05-0.1 M NaCl. Electron microscopic studies employing the rotary shadowing technique indicate that 31-K protein particles bind to the ends of collagen molecules. We propose that this glycoprotein serves as anchorage site for extracellular collagen to the chondrocyte membrane and thus may be involved in cell-matrix interactions in cartilage. 相似文献
6.
A monoclonal antibody, CG4, was raised to a novel 60 kDa metalloproteinase purified from a bovine brain myelin glycoprotein fraction. Glycoproteins extracted from both myelin and nine different bovine tissues showed the 60 kDa CG4-immunoreactive band by immunoblotting in amounts that broadly paralleled enzymic activity of this metalloproteinase and varied relatively little among the tissues. 相似文献
7.
The enzyme dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130-fold before it was further purified by native and SDS gel chromatography. A 92-kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92-kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltransferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino-terminal and carboxy-terminal neighbours, respectively. 相似文献
8.
Purification and characterization of an 82-kD membrane protein as a neurite outgrowth factor binding protein: possible involvement of NOF binding protein in axonal outgrowth in developing retina 总被引:1,自引:0,他引:1
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《The Journal of cell biology》1991,112(2):313-322
Neurite outgrowth factor (NOF) is a glycoprotein isolated from an extract of gizzard that induces neurite outgrowth from cultured retinal or ciliary ganglionic (CG) neurons. We have reported that a glycoprotein of approximately 82 kD solubilized from gizzard muscles binds to NOF (ligand blotting) and inhibits the neurite promoting activity of NOF (inhibition assay). The 82-kD protein (NOF binding protein) was purified from gizzard muscle membranes as a doublet band on SDS-PAGE and a polyclonal antibody was raised against it. An NOF binding protein in developing retina exhibited the same physicochemical properties as that of the gizzard muscle. Quantitative decrease in NOF binding protein in embryonic retinas was observed after day 11 by the inhibition assay, ligand blotting, and immunoblotting, its decrease being parallel with reduction of NOF-induced neurite outgrowth of embryonic retinas. In an immunohistochemical study, the antibody stained only the optic fiber layers of the retinas of 8-d embryos, and this staining was no longer detectable in retinas of 18-d embryos. These results suggest that the 82-kD protein is a novel membrane protein that behaves as an NOF receptor and that the loss of neuritic response of the retinal neurons to NOF reflects a decrease in NOF receptor molecules. 相似文献
9.
Characterization of a novel glycoprotein isolated from the basement membrane matrix of the Engelbreth-Holm-Swarm tumor 总被引:1,自引:0,他引:1
L K Robinson V A Murrah M P Moyer D H Rohrbach 《The Journal of biological chemistry》1989,264(9):5141-5147
A previously undescribed protein has been isolated and purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor, a murine tumor that synthesizes an extensive matrix composed of basement membrane molecules. Molecular characterization of the molecule determined that it is a glycoprotein with internal disulfide bonds and an isoelectric point of 6.0. Electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the glycoprotein migrated as a diffuse band with a molecular weight of approximately 72,000-80,000. The amino acid composition was significantly different from known basement membrane components. Polyclonal antibodies that specifically recognize the glycoprotein localized it to the kidney glomerular basement membrane. These antibodies did not cross-react with either known basement membrane components (laminin, type IV collagen, and heparan sulfate proteoglycan), with 70K "culture shock" protein or with components of normal mouse serum (including mouse transferrin, albumin, or alpha-fetoprotein), when analyzed by "Western" immunoblots. Our data indicate that the glycoprotein is synthesized by the EHS tumor cells and is present at relatively high levels in the EHS tumor matrix. 相似文献
10.
A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities. 相似文献
11.
A low molecular weight protein from dry mung bean (Vigna radiata) embryonic axes has been purified to near homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. It shows a molecular weight of about 12,000 in sodium dodecyl sulfate-polyacrylamide gels and a sedimentation coefficient of about 2 S in sucrose gradients. This protein occurs in greater amounts in dry axes than in dry cotyledons, and it dramatically disappears during early germination of the seed. Affinity chromatography tests do not indicate it as a trypsin inhibitor or as a glycoprotein. It is a water-soluble cytoplasmic protein exhibiting an amino acid composition characteristic of storage proteins with a high content of glutamic acid/glutamine. We suggest that it is a low molecular weight storage albumin.Abbreviations Asx
aspartic acid/asparagine
- BSA
bovine serum albumin
- Con A
concanavalin A
- EB
extraction buffer
- Glx
glutamic acid/glutamine
- HA
hydroxylapatite
- PB
phosphate buffer
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid 相似文献
12.
A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of 3H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10(-8)M; Dynorphin 1-8, 3.03 x 10(-7); U50,488H, 4.48 x 10(-9); U69-593,2.28 x 10(-8), morphine, 4.05 x 10(-6) DADLE, 6.47 x 10(-6) and naloxone, 2.64 x 10(-8). The purified receptor binds 8 nmole of 3H-Etorphine and 1.7 nmole 3H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed. 相似文献
13.
We have partially purified a lymphokine, costimulator, which is necessary to induce mitogenesis in mouse thymocytes in vitro. Costimulator is released from mouse leukocytes exposed to Con A for 12 to 18 hr. It has been purified more than 100 X by gel exclusion chromatography and isoelectric focusing. Thymocytes from CBA/J mice respond to the mitogenic lectin Con A only if the costimulator concentration is above a certain level. Culturing such cells with Con A at a density below 1 X 10(6) cells/ml produces costimulator concentrations too low for mitogenesis. This system has been developed into a quantitative assay for costimulator, to monitor purification, recovery, and biologic activity in various methods of molecular characterization. The activity is trypsin sensitive, and has a buoyant density characteristic of protein or glycoprotein. However, for a protein, it is relatively heat stable. Its m.w., established by carrying out sedimentation, gel filtration, and buoyant density measurements, is 30,500, and its frictional coefficient is 1.45. Costimulator purified by isoelectric focusing is active at 10(-10) M or lower in tissue culture. 相似文献
14.
Abstract— A low molecular weight glycoprotein has been demonstrated in myelin isolated from immature rat brains. Both short term and long term fucose incorporation studies have identified this protein in the proteolipid protein region of a sodium dodecyl sulfate, polyacrylamide gel. A 1.7-2.1 fold increase in radioactive fucose in this glycoprotein relative to the major myelin glycoprotein was seen after long term fucose incorporation (21 days) compared to short term incorporation (18–22 h). The demonstration that this fucose-labelled protein is distinguishable from that of proteolipid protein was achieved by a variety of independent techniques. One technique involved a comparison of ether-ethanol extracted, freshly isolated, myelin with myelin extracted with chloroform-methanol. Treatment of isolated myelin with chloroform-methanol results in the solubilization of the proteolopid protein and its subsequent absence on gel electrophoresis while, in contrast, an enhancement of fucose label was observed in the same region of the polyacrylamide gel. Another procedure involved the electrophoretic separation of the radioactive fucose peak from that of proteolipid protein by employing a continuous, phosphate buffered, gel system. Finally carbohydrate analysis by gas-liquid chromatography of a partially purified proteolipid protein fraction did not reveal significant amounts of carbohydrates which are characteristic of glycoproteins. The identification of this minor glycoprotein comigrating with proteolipid protein indicates, therefore, a greater complexity associated with the purified myelin membrane than has been previously demonstrated. 相似文献
15.
C H Chien S C Chang Y H Wei 《Proceedings of the National Science Council, Republic of China. Part B, Life sciences》1989,13(3):201-209
Neuraminidase (EC 3.2.1.18) has been purified from the culture medium of Clostridium perfringens ATCC 10543, through steps of gel filtration on Sephadex G-75 column, DEAE-cellulose DE 23 anion exchange chromatography, and isochromatofocusing. A homogeneous enzyme was obtained with a 7552-fold increase in specific activity to 295 units/mg protein. The yield was about 25%. The enzyme consists of a single polypeptide with a molecular weight of 69,000 as determined by SDS-polyacrylamide gel electrophoresis. Kinetic studies showed that Km is 1.5 mM for sialyllactose and Vmax is 0.41 mumole/min/ml at the enzyme concentration of 0.14 microgram/ml. The enzyme is stable at pH 5.2-8.0 with an optimal pH of 6.0. A concentrated solution of the purified enzyme was stable over one year at 4 degrees C. The purified enzyme hydrolyzed human alpha 1-acid glycoprotein completely; thus, it can be used in the clinical assay of N-acetylneuraminic acid in the serum. 相似文献
16.
C S Cierniewski S Niewiarowski D Hershock B Rucinski A H Schmaier 《Biochimica et biophysica acta》1987,924(1):216-224
Glycoprotein IIIa was quantitated in human platelets by radioimmunoassay using antisera specific to platelet membranes and purified glycoprotein IIIa. Glycoprotein IIIa and glycoprotein IIb were isolated from washed platelets by Triton X-114 extraction followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioiodinated glycoprotein IIIa was further purified by affinity chromatography on Lentil lectin-Sepharose 4B. Purified glycoprotein IIb showed little crossreactivity with 125I-labeled glycoprotein IIIa using the anti-platelet membrane or anti-glycoprotein IIIa antisera on a competition inhibition radioimmunoassay. The expression of glycoprotein IIIa epitopes were the same for the purified glycoprotein IIIa and glycoprotein IIIa in Triton X-100 solubilized platelets. A 66 kDa protein derived from glycoprotein IIIa by limited proteolysis of platelet membranes also expressed the same epitopes as intact glycoprotein IIIa. Solubilized platelets contained approximately 16 micrograms of total glycoprotein IIIa antigen per 10(9) cells. The level of glycoprotein IIIa determined by radioimmunoassay in one patient with Glanzmann's thrombasthenia amounted to 6.7% of normal and it was close to the values obtained by other methods. 相似文献
17.
C S Bambra 《Journal of reproduction and fertility》1987,79(2):421-430
Baboon CG from the urine and placenta (Days 33-45 of gestation) was purified by ammonium acetate/alcohol extraction followed by ionic exchange, gel filtration and affinity chromatography. The CG activity was monitored using a double-antibody radioimmunoassay utilizing anti-baboon CG and hCG both as the labelled ligand and standard. Biological activity was measured by the rat luteal cell radioreceptor assay. The purified preparation exhibited heterogeneity in terms of its behaviour during ionic exchange chromatography and isoelectric focussing. Like hCG, baboon CG was made up of two non-covalently linked subunits: the Stokes' radii were 36.5, 29.0 and 22.0 X 10(-10) m for native CG, the beta subunit and the alpha subunit. The material focussed between pH 4.2 and 5.5. Relative to the second international hCG standard the biological potency of the purified urinary baboon CG was 4058 i.u. whilst the immunological activity was 4364 i.u. It is concluded that baboon and human CG are very similar with respect to their physicochemical properties and that baboon CG can be purified by the methods that have been developed for hCG. 相似文献
18.
Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C. 总被引:1,自引:0,他引:1
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A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen. 相似文献
19.
It is well established that the LH/CG receptor expressed in gonadal cells is an 85- to 92-kilodalton (kDa) glycoprotein. Additionally, however, a number of reports have noted the existence of other putative receptor species, but few attempts have been made to characterize these variant receptor species. A cell line [293L(wt1)] had previously been isolated which expresses large numbers of high affinity cell surface LH/CG receptors. Visualization of the LH/CG receptor species expressed in these cells and in rat luteal cells using ligand blots revealed 85- and 90-kDa LH/CG receptors, respectively, while immunoblots revealed another 68-kDa glycoprotein receptor in both cell types. The presence of both the 85- and 68-kDa receptor species was confirmed using immunoprecipitation and affinity purification of metabolically labeled 293L(wt1) cells. Enzymatic deglycosylations established that the 85-kDa receptor is a sialoprotein, while the 68-kDa species contains exposed high mannose residues. Protease digestion before LH/CG receptor immunoprecipitations localized the 85-kDa receptor on the plasma membrane, while the 68-kDa receptor was shown to be located intracellularly. Pulse-chase experiments were then used to positively establish that the 68-kDa receptor protein is actually a precursor of the 85-kDa LH/CG receptor species. 相似文献
20.
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