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1.
A short-term incubation system was used to study proteoglycan synthesis during the early stages of medullary bone formation in estrogen-treated male Japanese quail. The proteoglycans were separated by chromatography on a DEAE Bio-Gel A column eluted with a 400-ml 0-1 M NaCl gradient. The profile from uninjected control birds showed a single peak, whereas profiles from estrogen-treated birds showed development of another peak. Incorporation of [35S]sulfate into the estrogen-induced proteoglycan increased most dramatically between 25 and 37 h after hormone treatment. The estrogen-induced proteoglycan has a Kav = 0.65 on Sepharose CL-4B, an average buoyant density of 1.50 g/ml, and contains keratan sulfate as its constituent glycosaminoglycan. The second proteoglycan has a Kav = 0.52 on Sepharose CL-4B, an average buoyant density of greater than or equal to 1.7 g/ml, and has chondroitin sulfate as it major glycosaminoglycan. It may also contain some heparin or heparan sulfate. The results support the usefulness of the incubation system for studying the dynamics of bone matrix production. 相似文献
2.
S. Fournier-Delpech M. Courot 《Biochemical and biophysical research communications》1980,96(2):756-761
Con A Receptors from the sperm plasma membrane were quantitated (using 3H acetyl-Con A) along the epididymal duct; they diminished in the second part of the epididymis as compared to the epididymal head. Glycoproteins having affinity for Con A were partially characterized: washed spermatozoa from rete testis (= testicular spermatozoa), middle corpus and distal cauda epididymis were labelled (125I Na). Proteins of their plasma membrane were extracted (Triton ×100, 0.1% and chromatography affinity): differences appeared in ACA44 profiles from 125I Con A Glycoprotein extractions between testicular spermatozoa (2 major peaks Kav= 0.41 and 0.52) and epididymal spermatozoa (3 major peaks Kav= 0.33–0.34, 0.41 and 0.52 and additional minor peaks between 0.66 and 1.00). The peak Kav= 0.41 diminished considerably on epididymal spermatozoa as compared to testicular spermatozoa. 相似文献
3.
Pancreatic polypeptide (PP) is synthesized as an amino-terminal moiety of a precursor peptide and is released into plasma during stimulation as an amidated hormone (PP1-36). The purpose of this investigation was to ascertain the immunoreactive forms of PP in human plasma using HPLC chromatographic technique. Plasma was obtained from five normal volunteers under various postprandial intervals and from the Blood Bank. PP in each plasma sample was processed for HPLC analysis by immunoprecipitation and/or immunoaffinity extractions. Migration patterns of PP-forms were identified under isocratic elution. This study shows that human plasma contains four distinct immunoreactive (IR) forms of PP during stimulation by a protein-rich meal. These forms are PP1-36 (peak 4), PP3-36 (peak 3) and unidentified material migrating as peak 2 and peak 1. The corresponding migration constants were Kav 0.828 +/- 0.04, Kav 0.790 +/- 0.003, Kav 0.570 +/- 0.009 and Kav 0.409 +/- 0.007, respectively. The predominant fasting from of IR PP chromatographed as peak 1, while peaks 2 and 4 were reduced in amplitude. The 1 h and 3 h postprandial chromatograms of HPLC profiles of plasma PP were similar in shape but lower in relative magnitude and amplitude. The authenticity of peak 4 as the migration of native PP1-36 was confirmed using purified IR native PP1-36 extracted from human pancreas. Partial amino acid sequence analysis of PP peak 3 revealed deletions of two N-terminal amino acid residues. The chemical identities of peaks 1 and 2 are unknown but appear to differ from PP in peaks 3 and 4 by virtue of their migration profiles. It is concluded that there are at least four distinct IR forms of PP in human plasma. Native PP1-36 accounts for less than 1% of total PP after an overnight fast and is about 1/3 of total postprandial IR plasma PP. Discernment of the nature and etiology of forms of PP in plasma may provide a new understanding of the role of PP in mammalian physiology. 相似文献
4.
Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts. 总被引:7,自引:0,他引:7
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S Johansson K Hedman L Kjelln J Christner A Vaheri M Hk 《The Biochemical journal》1985,232(1):161-168
Subconfluent cultures of human embryonic skin fibroblasts were labelled with [35S]sulphate for 3 days, after which cell-free extracellular matrix was isolated. A chondroitin sulphate proteoglycan (CSPG) and a heparan sulphate proteoglycan (HSPG) were purified from the matrix. Chromatography on Sepharose CL-2B gave peak Kav. values of 0.35 and 0.38 respectively for the CSPG and the HSPG. The polysaccharide chains released from the two PGs were of similar size (Kav. 0.50 on Sepharose CL-4B). Approx. 50% of the CSPG showed affinity for hyaluronic acid (HA). However, it differed immunologically from the HA-aggregating CSPG of human articular cartilage, and had a larger core protein (apparent molecular mass 290 kDa) than had the cartilage PG. Neither metabolically [35S]sulphate-labelled PGs, isolated from the medium of fibroblast cultures, nor chemically 3H-labelled polysaccharides (HA, CS, HS and heparin) were incorporated into the extracellular matrix when added to unlabelled cell cultures. These results indicate that the matrix PGs are not derived from the PGs present in the medium and that an interation between polysaccharide chains and matrix components is not sufficient for incorporation of PGs into the matrix. Incubation of cell-free 35S-labelled matrix with unlabelled polysaccharides did not lead to the release of any 35S-labelled material, supporting this conclusion. Furthermore, so-called 'link proteins' were not present in the fibroblast cultures, indicating that the CSPGs were anchored in the matrix in a manner different from the link-stabilized association of CSPG with HA in chondrocyte matrix. The identification of a proteinase, secreted by fibroblasts in culture, that after activation with heparin has the ability to release 35S-labelled PGs from the matrix may also indicate that the core proteins are important for the association of the PGs to the matrix. 相似文献
5.
W E Serafin H R Katz K F Austen R L Stevens 《The Journal of biological chemistry》1986,261(32):15017-15021
The predominant [3H]diisopropyl fluorophosphate (DFP)-binding proteins that are released from the secretory granules of activated mouse bone marrow-derived mast cells (BMMC) are demonstrated to have an isoelectric point of approximately 9.1 and to be complexed to proteoglycans. Upon Sepharose CL-2B chromatography of the supernatants of calcium ionophore-activated BMMC, 67-78% of the total exocytosed [3H]DFP-binding proteins co-eluted in the excluded volume of the column as a greater than 1 X 10(7) Mr complex bound to 4-7% of the total exocytosed proteoglycans. The remainder of the exocytosed proteoglycans, which filtered in the included volume of the gel filtration column with a Kav of 0.66, contained chondroitin sulfate E glycosaminoglycans. After dissociation of the large Mr complexes of [3H]DFP-binding proteins-proteoglycans with 5 M NaCl and removal of the proteins via phenyl-Sepharose chromatography, the proteoglycans filtered from the Sepharose CL-2B column as a single peak with a Kav of 0.66. The susceptibility of 24-59% and 36-76% of the glycosaminoglycans in the large Mr complex to degradation by nitrous acid and chondroitinase ABC, respectively, indicated the presence of proteoglycans that contained heparin and chondroitin sulfate glycosaminoglycans. Disaccharide analysis revealed that the chondroitin sulfate in the high Mr complex was chondroitin sulfate E. Following chondroitinase ABC treatment of the large Mr complex, the residual heparin proteoglycans filtered on Sepharose CL-4B under dissociative conditions with the same Kav as the original, untreated proteoglycans. Thus, the protein-proteoglycan complexes that are exocytosed from activated mouse BMMC contain approximately equal amounts of proteoglycans of comparable size that bear either predominantly heparin or predominantly chondroitin sulfate E glycosaminoglycans. The demonstration of these secreted complexes indicates that the intragranular protease-resistant heparin and chondroitin sulfate E proteoglycans in the T cell factor-dependent BMMC bind serine proteases throughout the activation-secretion response. 相似文献
6.
Cell surface phosphatidylinositol-anchored heparan sulfate proteoglycan initiates mouse melanoma cell adhesion to a fibronectin-derived, heparin-binding synthetic peptide 总被引:4,自引:0,他引:4
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S L Drake D J Klein D J Mickelson T R Oegema L T Furcht J B McCarthy 《The Journal of cell biology》1992,117(6):1331-1341
Cell surface heparan sulfate proteoglycan (HSPG) from metastatic mouse melanoma cells initiates cell adhesion to the synthetic peptide FN-C/H II, a heparin-binding peptide from the 33-kD A chain-derived fragment of fibronectin. Mouse melanoma cell adhesion to FN-C/H II was sensitive to soluble heparin and pretreatment of mouse melanoma cells with heparitinase. In contrast, cell adhesion to the fibronectin synthetic peptide CS1 is mediated through an alpha 4 beta 1 integrin and was resistant to heparin or heparitinase treatment. Mouse melanoma cell HSPG was metabolically labeled with [35S]sulfate and extracted with detergent. After HPLC-DEAE purification, 35S-HSPG eluted from a dissociative CL-4B column with a Kav approximately 0.45, while 35S-heparan sulfate (HS) chains eluted with a Kav approximately 0.62. The HSPG contained a major 63-kD core protein after heparitinase digestion. Polyclonal antibodies generated against HSPG purified from mouse melanoma cells grown in vivo also identified a 63-kD core protein. This HSPG is an integral plasma membrane component by virtue of its binding to Octyl Sepharose affinity columns and that anti-HSPG antibody staining exhibited a cell surface localization. The HSPG is anchored to the cell surface through phosphatidylinositol (PI) linkages, as evidenced in part by the ability of PI-specific phospholipase C to eliminate binding of the detergent-extracted HSPG to Octyl Sepharose. Furthermore, the mouse melanoma HSPG core protein could be metabolically labeled with 3H-ethanolamine. The involvement of mouse melanoma cell surface HSPG in cell adhesion to fibronectin was also demonstrated by the ability of anti-HSPG antibodies and anti-HSPG IgG Fab monomers to inhibit mouse melanoma cell adhesion to FN-C/H II. 35S-HSPG and 35S-HS bind to FN-C/H II affinity columns and require 0.25 M NaCl for elution. However, heparitinase-treated 125I-labeled HSPG failed to bind FN-C/H II, suggesting that HS, and not HSPG core protein, binds FN-C/H II. These data support the hypothesis that a phosphatidylinositol-anchored HSPG on mouse melanoma cells (MPIHP-63) initiates recognition to FN-C/H II, and implicate PI-associated signal transduction pathways in mediating melanoma cell adhesion to this defined ligand. 相似文献
7.
This study has examined changes in proteoglycan synthesis during megakaryocyte maturation in vivo. Guinea pigs were injected with Na235SO4, and megakaryocytes and platelets were isolated from 3 h to 5 days later. The proteoglycans and other sulfated molecules in both cells were characterized at each time point by gel filtration, ion-exchange chromatography, gel electrophoresis, and chemical and enzymatic digestions. Two populations of chondroitin 6-sulfate proteoglycans were found by DEAE-Sephacel chromatography. The major fraction was eluted with 4 M guanidine hydrochloride and the minor fraction with 4 M guanidine HCl, 2% Triton X-100. The Kav of the major proteoglycan peak in the platelets at 1 day after injection was 0.18-0.20 on Sepharose CL-6B and decreased gradually to 0.12 by 3 days, when proteoglycan radioactivity per cell was maximal. The peak for megakaryocyte proteoglycans at 3 h was broad, with Kav = 0.1-0.2. The appearance of different portions of the proteoglycan peak in platelets coincided with their disappearance from megakaryocytes. Proteoglycan size was a function of glycosaminoglycan chain length. The proteoglycans eluted with Triton X-100 from DEAE-Sephacel (Kav = 0.04-0.07 on Sepharose CL-6B) were not labeled in platelets until 2 days after injection. Our data suggest that megakaryocytes synthesize different-sized chondroitin sulfate proteoglycans at different stages of development. The proteoglycans of the major fraction were released from platelets in response to thrombin, and a small amount was released by ADP. The proteoglycans of the Triton X-100 eluate were not released by thrombin or ADP. About 20% of the sulfate radioactivity was incorporated into molecules that appear to be sulfated proteins and were not released by thrombin or ADP. 相似文献
8.
Selenocysteine-containing proteins from rat and monkey plasma 总被引:1,自引:0,他引:1
This investigation was carried out to determine whether a selenium-containing plasma protein in rat and monkey (Macaca mulata) plasma might be involved in selenium transport. Injection of [75Se]selenite or [75Se]selenomethionine was used to label a plasma protein. The native molecular weight of the protein from rat and monkey plasma was determined by gel filtration to be about 80 000. The molecular weight of a selenium-containing polypeptide prepared from the protein was about 45 000, as determined by gel filtration in the presence of sodium dodecyl sulfate. Selenium was attached to both the rat and monkey plasma protein in the form of the amino acid selenocysteine. The proportion of plasma selenium normally bound to the rat protein in vivo was less than 5%, and the half-life of selenium bound to the protein was a few hours. These findings are consistent with a selenium-transport function for this protein. 相似文献
9.
L S Forsberg S C Lazarus N Seno R DeVinney G H Caughey W M Gold 《Biochimica et biophysica acta》1988,967(3):416-428
The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin. 相似文献
10.
The flowering of interferon 总被引:2,自引:0,他引:2
D C Burke 《Biochimica et biophysica acta》1982,695(1):1-4
A microsomal vesicle fraction was prepared from rat liver homogenate by centrifugation in gradients of Percoll. The microsomes were subjected to gel filtration on Sephacryl S-1000 Superfine, which resolved the microsomes from Percoll. The elution pattern of the microsomal marker enzyme NADPH-cytochrome c reductase showed that the main part of the enzyme was present in a peak at Kav about 0.1, while Percoll eluted in a broad peak at Kav about 0.7. The total yield of eluted enzyme activity was 85%. The gel filtration had to be carried out in the presence of 10 mM tris or NaCl. At lower ionic strength or in 0.25 M sucrose alone, anomalous behaviour of the Percoll particles and microsomes on the gel was observed. Electron microscopy of samples from the void volume fraction of the Sephacryl S-1000 Superfine column showed an almost complete removal of Percoll from the microsomes. Furthermore, the vesicle preparation was essentially free of membrane fragments. 相似文献
11.
Identification of chondroitin sulfate E in human lung mast cells 总被引:3,自引:0,他引:3
H L Thompson E S Schulman D D Metcalfe 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(8):2708-2713
Human lung mast cells (HLMC) enriched up to 99% purity by counter current elutriation and density gradient centrifugation were labeled with 35S-sulfate to determine cell-associated proteoglycans. The 35S-labeled proteoglycans were extracted by the addition of detergent and 4 M guanidine-HCl, and separated from unincorporated precursor by Sephadex G-50 chromatography. 35S-Proteoglycans chromatographed over Sepharose 4B with a Kav of 0.48. 35S-Glycosaminoglycans separated from the parent 35S-proteoglycans by beta-elimination and chromatographed over Sepharose 4B with a Kav of 0.63. Characterization of 35S-proteoglycans by chondroitin ABC lyase treatment revealed approximately 36% of the proteoglycan to be composed of chondroitin sulfates. Analysis by HPLC of component disaccharides liberated by chondroitin ABC lyase using an amino-cyano-substituted silica column indicated that the chondroitin sulfates consisted of the monosulfated A disaccharide (GlcUA----GaINAc4SO4) (75%) and the over-sulfated E disaccharide (GlcUA----GaINAc4,6-diSO4) (25%). Nitrous acid/heparinase-susceptible heparin proteoglycans accounted for approximately 62% of the total 35S-proteoglycans present in the HLMC. Proteoglycans remaining after exposure of the original proteoglycan extract to either heparinase or chondroitin ABC lyase were of similar size, suggesting that the majority of heparin and chondroitin sulfate glycosaminoglycans were on separate protein cores. Proteoglycans extracted from HLMC were protease insensitive. Hence, in addition to heparin proteoglycans, HLMC synthesize a hitherto unrecognized quantity of chondroitin sulfate E proteoglycans. 相似文献
12.
K C Ehrlich 《The Journal of biological chemistry》1981,256(1):73-80
Cells having a fibroblast-like morphology were cultured from explants of adult rat lung tissue. (35S)Sulfate was incorporated into sulfated proteoglycans in the medium at a linear rate for up to 96 h, while the rate of incorporation into the cell layer increased gradually until reaching a plateau at 40 h. The culture medium contained proteoglycans which migrated as a single peak with Kav = 0.10 on Bio-Gel A-15. Their glycosaminoglycan components (Kav = 0.70 on Bio-Gel A-15) contained predominantly chondroitin sulfate (33 to 44% of the total galactosaminoglycans) or dermatan sulfate chains. Based on the results of chondroitinase AC-II and periodate degradation, disaccharide repeating units of the dermatan sulfate were composed of 36% iduronic acid, 50% 2-sulfoiduronate, and 14% glucuronic acid. A similar composition was found for the dermatan sulfate in the cell fraction. Almost one-half of the sulfate label in the cell fraction was in a heparan sulfate proteoglycan which migrated on Bio-Gel A-15 with Kav = 0.30. The heparan sulfate chains (Kav = 0.81 on Bio-Gel A-15) had few, if any, sulfated N-acetylglucosamine residues and did not contain 2-sulfoiduronic acid in neighboring disaccharide repeat sequences. These results indicate that fibroblast-like lung cells synthesize several types of multichain sulfated proteoglycans which have properties in common with those found in lung tissues. 相似文献
13.
Human urine contains a hitherto unrecognized heparin-dependent inhibitor of activated protein C (APC) (Mr approximately 50,000) that coelutes from heparin-Sepharose together with the only observed peak of urokinase inhibitory activity at a position (0.35 M NaCl) similar to that of plasma protein C (PC) inhibitor. Based on functional assays and immunoblot studies, urokinase and APC compete for this crude inhibitor in the absence or presence of heparin. These results suggest that the same heparin-dependent urinary inhibitor that is immunologically different from several known protease inhibitors is responsible for the observed inhibition of APC and urokinase. In the absence of heparin this inhibitor inhibits APC and urokinase with similar rates, and heparin enhances its inhibitory activity toward both enzymes with more pronounced stimulation of its PC inhibitory activity than its urokinase inhibitory activity. Half-maximal stimulation of inhibition of APC occurs at about 2 mU/ml and maximal stimulation (approximately 10-fold increase of the pseudo-first-order rate constant) at greater than or equal to 50 mU/ml of heparin. This is the first demonstration of competition between APC and urokinase for a heparin-dependent inhibitor. These results may therefore represent a new link between the two major antithrombotic pathways, the PC pathway and the fibrinolytic system. 相似文献
14.
Inhibition of platelet function by cAMP is due at least in part to a reduction in the agonist stimulated increase in cytoplasmic calcium during cell activation. This inhibition is also associated with cAMP-dependent phosphorylation of thrombolamban, a 22 kDa phosphoprotein which is present in the same membrane fraction as the calcium-dependent ATPase. Phosphorylation of this protein has been correlated with increased uptake of calcium by microsomal membranes. The present study was undertaken to examine the interaction of thrombolamban with the Ca++-ATPase in order to assess the possibility that the increased calcium uptake was by a direct effect of thrombolamban on Ca++-ATPase activity or that thrombolamban was a component of the Ca++-ATPase. Several approaches were utilized to assess the interaction of thrombolamban with the microsomal Ca++-ATPase. Gel filtration of labeled microsomes solubilized under non-denaturing conditions showed a major peak of radioactivity (Kav 0.64) corresponding to thrombolamban which was well separated from the Ca++-ATPase activity (Kav 0.09). Chemical cross-linking studies using partially purified thrombolamban and intact microsomes showed incorporation of the phosphoprotein into a 147,000 dalton complex. Indirect immunostaining with an anti-Ca++-ATPase antibody failed to demonstrate the Ca++-ATPase in the 147,000 dalton complex. Recombination of the phosphorylated thrombolamban with the Ca++-ATPase had no effect on Ca++-ATPase activity. These results indicate that, under the conditions used in these experiments, there was no apparent interaction between thrombolamban and the microsomal Ca++-ATPase. We conclude that thrombolamban is covalently bound to the Ca++-ATPase. 相似文献
15.
Previously, a selenium-containing protein with subunit molecular weight of 15 kDa was found in peripheral human granulocytes.
In continuation of this work, the present communication accounts for purification, identification, and characterization of
this major selenium-containing protein. The protein was purified on a heparin-Sepharose column followed by Sephacryl S-200
column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis visualized two bands with
subunit molecular weights around 15 kDa.o-Phthaldialdehyde precolumn derivatization and reverse-phase high-performance liquid chromatography showed that the protein
contains selenocysteine or selenocystine residues. Highperformance gel filtration and isoelectric focusing revealed that the
protein had an apparent molecular weight of 32 kDa and apI value of 7.9. The addition of the protein synthesis inhibitor puromycin to the cell culture medium decreased the 15-kDa protein
synthesis. These data suggest that the major selenium-containing protein in peripheral human granulocytes might be a protein
with two subunits around 15 kDa. Enzyme studies showed that the protein had peroxidase activity assayed with H2O2 as a substrate and O-dianisidine as a hydrogen donor. This enzymatic activity competed with glutathione peroxidase on the
consumption of H2O2, leading to an “inhibiton” of glutathione peroxidase (GSH-Px) activity. Sodium azide could eliminate the inhibition of the
protein to GSH-Px. All of the above results implicated that the protein might be a H2O2-dependent seleniumcontaining peroxidase different from GSH-Px. Therefore, the biological function of the protein could be
related to eliminating H2O2 generated in the respiratory burst reaction of granulocytes, thus protecting these cells from oxida-tive damage during phagocytosis. 相似文献
16.
Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products. 相似文献
17.
Jacobson GA Featherstone AM Townsend AT Lord R Peterson GM 《Biological trace element research》2005,107(3):213-220
Several recent analytical methods for determination of Se and selenoprotein P have involved high-performance liquid chromatography (HPLC) using heparin-affinity columns coupled to inductively coupled plasma-mass spectrometry (ICP-MS) for Se detection. HPLC-ICP-MS chromatography using tandem HPLC columns with ICP-MS detection was used to detect the major selenium-containing proteins in plasma (glutathione peroxidase, albumin, and selenoprotein P). The efficiency of HPLC separation of plasma selenoprotein P was investigated by analyzing HPLC fractions using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblot analysis. The HPLC fraction corresponding to selenoprotein P contained 25.1% of total selenoprotein P as measured by immunoblot analysis. The majority (74.9%) of total selenoprotein P found by immunoblot analysis was contained in the early HPLC fractions, consistent with either poor heparin affinity, which was not evident based on the HPLC-ICP-MS technique alone or nonspecific binding of the antibody. Immunoblot analysis of selenoprotein relies on antibodies binding to a selenoprotein P epitope, which might be preserved when selenoprotein P is broken down to release selenocysteine residues. Immunoblot methods overestimate selenoprotein P and are not suitable for determinations of intact selenoprotein P. 相似文献
18.
Ritsuko Katoh-Semba Atsuhiko Oohira Shigeo Kashiwamata 《Journal of neurochemistry》1992,59(1):282-289
Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC12 cells after 3 days of culture was in the range of 90-190 kDa for the intact form (Kav = 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90-190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120-150 kDa and 120-190 kDa (Kav = 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav = 0.22 on Sephacryl S-300 [40-84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
Competition between cholesterol and phosphatidylcholine for the hydrophobic surface of sarcoplasmic reticulum Ca2+-ATPase 总被引:1,自引:0,他引:1
A multiple equilibrium binding model is used to examine phospholipid and cholesterol binding with the transmembranous protein Ca2+-ATPase (calcium pump). The protein was reconstituted in egg phosphatidylcholine bilayers by lipid substitution of rabbit muscle sarcoplasmic reticulum. Electron spin resonance spectra of a phosphatidylcholine spin-label and a recently developed cholesterol spin-label show two major spectral contributions, a motionally restricted component consistent with interactions between the label and the protein surface and another component characteristic of motion of the label in a fluid lipid bilayer. The number of lipid binding (or contact) sites at the hydrophobic surface of the protein is calculated to be N = 22 +/- 2. Experiments with intact sarcoplasmic reticulum membranes give approximately the same value for N. The relative binding constants are Kav approximately 1 for the phosphatidylcholine label and Kav approximately 0.65 for the cholesterol spin-label. Thus, cholesterol does contact the surface of the protein, but with a somewhat lower probability than phosphatidylcholine. This is confirmed by competition experiments where unlabeled cholesterol and the phospholipid spin-label are both present in the bilayer. Evidently the flexible acyl chains of the phospholipid molecules accommodate more readily to the irregular surface of the protein than does the rigid steroid structure of cholesterol. 相似文献
20.
The synthesis and secretion of chondroitin sulphate proteoglycan (CSPG) was examined in human muscle cultures during myogenesis prior to myoblast fusion and following myotube formation. Results from this study demonstrate that the major CSPG secreted into the medium had a Kav of 0.15 on Sephacryl 500 (exclusion limit of 10(7) Da) and contained predominantly unsulphated residues in mononucleated cell cultures but these became increasingly sulphated in postfusion cultures. Fibroblasts synthesised small amounts of a smaller molecular weight CSPG indicating that the Kav 0.15 proteoglycan is solely synthesised by cells of the myogenic lineage. These findings illustrate that sulphation of CSPG is developmentally regulated during myogenesis of human muscle cells grown under differentiating conditions. 相似文献